fphar-08-00291 May 22, 2017 Time: 17:37 # 1

REVIEWpublished: 23 May 2017

doi: 10.3389/fphar.2017.00291

Edited by:Richard Lowell Bell,

Indiana University School of Medicine,United States

Reviewed by:Thomas Grutter,

University of Strasbourg – CNRS,France

Daryl L. Davies,University of Southern California,

United States

*Correspondence:Leanne Stokes

l.stokes@uea.ac.ukSamuel J. Fountain

s.j.fountain@uea.ac.uk

Specialty section:This article was submitted to

Neuropharmacology,a section of the journal

Frontiers in Pharmacology

Received: 03 February 2017Accepted: 05 May 2017Published: 23 May 2017

Citation:Stokes L, Layhadi JA, Bibic L,

Dhuna K and Fountain SJ (2017)P2X4 Receptor Function

in the Nervous System and CurrentBreakthroughs in Pharmacology.

Front. Pharmacol. 8:291.doi: 10.3389/fphar.2017.00291

P2X4 Receptor Function in theNervous System and CurrentBreakthroughs in PharmacologyLeanne Stokes1,2*, Janice A. Layhadi3, Lucka Bibic1, Kshitija Dhuna2 andSamuel J. Fountain3*

1 School of Pharmacy, University of East Anglia, Norwich Research Park, Norwich, United Kingdom, 2 School of Biomedicaland Health Sciences, RMIT University, Bundoora, VIC, Australia, 3 Biomedical Research Centre, School of BiologicalSciences, University of East Anglia, Norwich, United Kingdom

Adenosine 5′-triphosphate is a well-known extracellular signaling molecule andneurotransmitter known to activate purinergic P2X receptors. Information has beenelucidated about the structure and gating of P2X channels following the determinationof the crystal structure of P2X4 (zebrafish), however, there is still much to discoverregarding the role of this receptor in the central nervous system (CNS). In thisreview we provide an overview of what is known about P2X4 expression in theCNS and discuss evidence for pathophysiological roles in neuroinflammation andneuropathic pain. Recent advances in the development of pharmacological toolsincluding selective antagonists (5-BDBD, PSB-12062, BX430) and positive modulators(ivermectin, avermectins, divalent cations) of P2X4 will be discussed.

Keywords: ATP, P2X4, microglia, pain, antagonist, ivermectin

INTRODUCTION

Purinergic P2X receptors are involved in mediating a multitude of functions in health anddisease via the actions of extracellular adenosine 5′-triphosphate (ATP) (North, 2002; Burnstockand Kennedy, 2011; Khakh and North, 2012). Although ATP has long been recognized asan intracellular energy source, its acceptance as an extracellular signaling molecule has takenconsiderably longer (Burnstock, 2006). It is now widely recognized that extracellular ATP acts aseither sole neurotransmitter or crucial co-neurotransmitter in most nerves in both the peripheralnervous system and central nervous system (CNS) (Burnstock, 2006; Khakh and North, 2012).For example, under physiological conditions, astrocytes and neurons are responsible for releasingsmall amounts of ATP, which can then act on purinergic receptors – either P2X ion channels orP2Y G protein-coupled receptors - expressed on both neurons and glial cells. In healthy tissues,ATP released from cells is tightly regulated with cell surface ectonucleotidases serving to terminatepurinergic signaling (Cardoso et al., 2015) (this is analogous to the activity of cholinesterases atcholinergic synapses). However, when released from injured cells, ATP can initiate inflammationand further amplify and sustain cell-mediated immunity through P2 receptors (Idzko et al., 2014).

P2X receptors are non-selective cation channels that open in response to ATP binding,allowing the rapid flow of ions (K+, Na+, Ca2+) across the membrane. Seven genes encodeP2X receptor subunits (P2X1-7) that are expressed in all tissues in humans and mice (North,2002; Khakh and North, 2006). P2X receptors share a common topology assembling as trimers(Hattori and Gouaux, 2012), with evidence of both homomeric and heteromeric assembly(Saul et al., 2013). Different subtypes are involved in specialized functions depending on their

Frontiers in Pharmacology | www.frontiersin.org 1 May 2017 | Volume 8 | Article 291

fphar-08-00291 May 22, 2017 Time: 17:37 # 2

Stokes et al. P2X4 Function and Pharmacology

distribution and biophysical properties (Surprenant and North,2009). These differences provide an opportunity for tissue-specific inhibition of one subtype without affecting thefunction of others. Such challenges represent a pharmacologicalopportunity to intervene in physiological processes includingmodulation of synaptic transmission, taste, pain sensation, andinflammation (Khakh and North, 2006).

Progress on several fronts has revealed the involvement ofP2X4 in a variety of pathophysiological processes. For example,P2X4 are implicated in neuropathic pain (Tsuda et al., 2003,2009a; Coull et al., 2005; Ulmann et al., 2008), inflammatorypain (Ulmann et al., 2010), epilepsy (Ulmann et al., 2013), lungsurfactant secretion (Miklavc et al., 2013), alcohol intake andpreference (Wyatt et al., 2014; Franklin et al., 2015), morphine-induced hyperalgesia (Ferrini et al., 2013), cardiac function(Yang et al., 2014) and P2X4 deficiency is associated withsociocommunicative impairments (Wyatt et al., 2013) and alteredflow-dependent blood vessel remodeling (Yamamoto et al., 2006).However, the study of P2X4 has been seriously hampered by anabsence of selective pharmacological tools. While the discovery ofselective antagonist molecules against P2X4 would facilitate thefurther elucidation of their physiological roles, their applicationmight also hold promises to lead us into novel therapeutics forhuman diseases. Here we aim to present a broad, comprehensiveoverview of P2X4 receptors, their function in the CNS, and theiremerging pharmacology.

P2X4; ION CHANNEL STRUCTURE ANDPERMEABILITY

The P2X4 receptor, now simply termed P2X4 using the latestIUPHAR nomenclature (Di Virgilio et al., 2015), is a ligand-gatedion channel belonging to the P2X receptor family. This familyof ion channels are activated by extracellular ATP and functionas non-selective cation channels permitting Na+, K+ and Ca2+

ion fluxes (North, 2002). The Ca2+ permeability of P2X4 isthe highest among the P2X family (Egan and Khakh, 2004).Measurement of the fractional calcium current (Pf%) throughP2X4 using the patch clamp photometry technique (measuringthe ATP-induced inward current and the concomitant decreasein the emission of fura-2 fluorescence excited at 380nm) yieldsvalues of 11% and 15% for rat P2X4 and human P2X4,respectively (Egan and Khakh, 2004).

P2X subunits are arranged into trimeric structures inthe plasma membrane, either in homomeric or heteromericformations (Saul et al., 2013). This trimeric organization of P2X4was confirmed in 2009 by solving the crystal structure of azebrafish P2X4 (zP2X4) in the closed state at 3.1A resolution(Kawate et al., 2009). Three inter-subunit binding sites for ATPwere defined in the P2X extracellular domain. In 2012 a crystalstructure for the ATP-bound state of zP2X4 was solved revealingthe open pore conformation of the channel (Hattori and Gouaux,2012). This also gave structural insight into the mechanicsof channel opening in response to ATP binding; fenestrationsopen up in close proximity to the plasma membrane providinglateral access pathways for cation entry to the ion channel

pore (Samways et al., 2011; Hattori and Gouaux, 2012; Gaoet al., 2015). This movement of the extracellular domains uponagonist binding leads to iris-like rotational movements of thetransmembrane domains lining the channel pore thus describingthe predicted activation mechanism of P2X4 channels (Hattoriand Gouaux, 2012). Fast-scanning atomic force microscopy hasalso been used to reveal the trimeric structure of P2X4 andmovement of subunits following ATP binding (Shinozaki et al.,2009).

The P2X4 subtype can heteromerically assemble with otherP2X members including P2X6, P2X7 and P2X1 (Le et al.,1998; Nicke et al., 2005; Antonio et al., 2011). These studieshave typically used immunoprecipitation of P2X4-containingprotein complexes and detection of other P2X using antibodiesin combination with altered functional and pharmacologicalproperties as major pieces of experimental evidence. For example,Xenopus oocytes expressing both P2X1 and P2X4 exhibit a slowlydesensitizing current similar to homomeric P2X4 but in thepresence of P2X1 there is significant activation by αβ-MeATPand inhibition by suramin (Nicke et al., 2005), properties notseen with either P2X4 or P2X1 alone. The reported interactionwith P2X7 is much more contentious. Evidence first suggestedthat P2X4 and P2X7 could form heterotrimeric complexes(Boumechache et al., 2009) but then further investigationsrevealed that homotrimers of P2X4 and P2X7 may interact witheach other (Antonio et al., 2011).

PERMEABILITY

Khakh and colleagues first described different permeability statesfor P2X4, denoting two states (I1 and I2 state) in response to asustained exposure to ATP (Khakh et al., 1999a). This secondarypermeability state (I2) displays increased NMDG+ permeationsuggestive of a larger channel pore size and a dynamic changein ion selectivity. This concept has recently been challenged andVrev changes recorded during NMDG+/Na+ bi-ionic conditionsis thought to be due to alterations in intracellular ion depletionand accumulation (Li et al., 2015). This data can be interpretedas lack of evidence for dynamic changes in ion conductance butwhether P2X channels are directly permeable to large cationssuch as NMDG+ still warrants further investigation. Furtherevidence for a secondary permeability state (or permeation oflarge organic cations) for P2X4 comes from the use of membraneimpermeant fluorescent dyes such as YOPRO-1 iodide andethidium bromide (Khakh et al., 1999a; Bernier et al., 2012).Whether these dyes permeate directly through the P2X4 channelor an associated conduit is not yet clear. Much evidence existsfor a large secondary permeation pathway activated by P2X7 innumerous cell types. Classically this is measured by the use of dyeuptake assays (in particular such as ethidium bromide, YOPRO-1 iodide, Lucifer yellow). Controversy surrounds the underlyingmechanism of action for P2X7 with some studies suggesting asecond protein may carry the dyes, such as pannexin-1 (Pelegrinand Surprenant, 2006) or anoctamin-6 (Ousingsawat et al., 2015)and others suggesting that P2X7 itself can allow passage of largemolecules (Browne et al., 2013). The physiological role of this

Frontiers in Pharmacology | www.frontiersin.org 2 May 2017 | Volume 8 | Article 291

fphar-08-00291 May 22, 2017 Time: 17:37 # 3

Stokes et al. P2X4 Function and Pharmacology

permeability pathway for large cations is still unclear for P2X7and P2X4 channels, and indeed those other ligand-gated ionchannels demonstrating a similar phenomenon (Chung et al.,2008; Banke et al., 2010).

EXPRESSION OF P2X4 IN THE CNS

P2X4 was originally cloned from rat brain cDNA in 1996 (Sotoet al., 1996a) and was the first P2X receptor detected in CNSneurons and blood vessels. P2X4 was subsequently cloned froma human brain sample (Garcia-Guzman et al., 1997) and wasalso found to exhibit a broad tissue expression pattern. Amongstthe P2X receptor subtypes, P2X4, along with P2X2 and P2X6,has been shown to be the most widespread and abundantlyexpressed functional ATP-gated purinergic receptor in the CNS,being found in most neurons and glial cells (Buell et al., 1996; Sotoet al., 1996a,b; Tsuda et al., 2003; Guo et al., 2005; Amadio et al.,2007; Vazquez-Villoldo et al., 2014). This has been illustratedat both mRNA and protein level by in situ hybridisation,immunohistochemistry, PCR and immunoblotting (Soto et al.,1996b; Lê et al., 1998; Bo et al., 2003).

NEURONS

Throughout the CNS, expression of P2X4 is widely observedin neurons. In the original paper cloning P2X4 from rat brain,Soto and colleagues demonstrated high levels of P2X4 mRNAin rat dentate gyrus granule cells, CA1/CA3 pyramidal cells,cerebellar cortex Purkinje cells, and neurons of the pontinenucleus (Soto et al., 1996a). Electron microscopy analysis suggestsP2X4 localisation in peri-synaptic regions of post-synapticterminals and on pre-synaptic terminals (Rubio and Soto, 2001).Immunohistochemistry also shows P2X4 to be expressed inGABAergic interneurons and GABAergic spiny neurons of therat striatum and substantia nigra (Amadio et al., 2007).

The hypothalamus and anterior pituitary gland abundantlyexpress P2X4 and this receptor may be involved in regulationof hypothalamo-pituitary functions in the CNS, as reviewedin Stojilkovic (2009). Immunohistochemistry studies identifiedP2X4 on paraventricular nucleus neurons projecting to therostral ventrolateral medulla and a potential role in regulatingsympathetic nerve activity (Cham et al., 2006). Paraventricularneurons, arcuate nucleus GnRH neurons and secretory cellsof the anterior pituitary all express P2X4 as illustratedthrough molecular biology techniques in combination withelectrophysiology (Zemkova et al., 2010). Functional P2X4receptors have also been identified in lactotrophs (He et al.,2003), and in the posterior pituitary system functional P2X4responses have been recorded from supraoptic neurons (Vavraet al., 2011; Stojilkovic and Zemkova, 2013). P2X4 has also beendemonstrated to be expressed in somatosensory cortical neurons(Lalo et al., 2007), nodose ganglion neurons (Tan et al., 2009),trigeminal neurons (Luo et al., 2006), vestibular ganglion neurons(Ito et al., 2010), retinal ganglion and bipolar cells (Wheeler-Schilling et al., 2001) and in spinal cord neurons (Bardoni et al.,1997; Kobayashi et al., 2005).

P2X4 has been implicated in physiological functions in theCNS including modulation of neurotransmission and synapticstrengthening (Rubio and Soto, 2001; Sim et al., 2006; Baxteret al., 2011). In the hippocampus P2X4 expression on pyramidalneurons is thought to contribute to synaptic plasticity and long-term potentiation (LTP). One of the initial studies that elaborateda role for P2X4 in LTP was performed in mice with a globaldeficiency in the p2rx4 gene (P2X4−/−) (Sim et al., 2006).Extracellular recording of field potentials from the CA1 region ofthe hippocampus in these P2X4-deficient mice revealed reducedsynaptic facilitation and induction of LTP compared to wild-type counterparts. In addition, ivermectin, a positive allostericmodulator of P2X4, increased LTP in wild-type mice but wasineffective in the P2X4−/− mice (Sim et al., 2006). This suggestedthat P2X4 contributes to strengthened synaptic activity duringLTP and it is hypothesized that calcium entry through sub-synaptic P2X4 contributes to synaptic strengthening by NMDAreceptor incorporation (Baxter et al., 2011).

Studies have also investigated cross-talk between P2X4 andother ion channels in neurons, in particular GABA(A) receptors(Jo et al., 2011) and nicotinic acetylcholine receptors (Khakhet al., 2000). In hypothalamic neurons increased expression ofP2X4 is associated with a reduction in GABAergic currents(Jo et al., 2011). There is some evidence for a physical couplingbetween P2X4 and GABA(A) receptors and this may play a rolein regulating synaptic signaling (Jo et al., 2011). A similar cross-talk has been demonstrated for P2X2 receptors and GABA(A)receptors and this cross-talk appears to be a general mechanismfor the regulation of GABAergic signaling, as reviewed inShrivastava et al. (2011). Therefore, P2X4 may be involvedin regulating excitatory vs. inhibitory neurotransmission inneurons, acting as a neuromodulator. The contribution of P2X4in this process will likely become clearer in the future withthe development of selective pharmacological tools and moreknockout mouse studies.

GLIAL CELLS

In the CNS P2X4 plays a role in modulating synaptic transmissionand communication between neurons and neighboring glial cells.Glia are the most abundant cell type accounting for >70% oftotal cells in the CNS and can be classified into three main types;astrocytes, oligodendrocytes and microglia.

The role of P2X4 in microglial cells has received muchattention in the last decade. Microglial cells, known as theresident macrophages in the CNS, originate from the yolk sacand are related to myeloid immune cells (Kettenmann et al.,2011; Saijo and Glass, 2011). Immunohistochemistry analysishas revealed abundant P2X4 reactivity on microglia withinthe brain and spinal cord (Tsuda et al., 2003; Ulmann et al.,2008). Despite the fact that P2X4 is abundantly expressed inmicroglial cells, the majority of labeled P2X4 appears to bepredominantly localized to intracellular lysosomal compartments(Qureshi et al., 2007; Toyomitsu et al., 2012). P2X4 has beensuggested to play a role in regulating the activation and migrationof microglial cells at sites of injury (Guo et al., 2005; Schwab

Frontiers in Pharmacology | www.frontiersin.org 3 May 2017 | Volume 8 | Article 291

fphar-08-00291 May 22, 2017 Time: 17:37 # 4

Stokes et al. P2X4 Function and Pharmacology

et al., 2005). Following peripheral nerve injury, the expressionof P2X4 is up-regulated in activated spinal cord microglia atthe transcriptional, translational and post-translational levels(Ulmann et al., 2008). Several factors that have been associatedwith regulating P2X4 expression include the chemokine CCL21(Biber et al., 2011), fibronectin (Nasu-Tada et al., 2006; Tsudaet al., 2008), and the cytokine interferon gamma (IFN-γ) (Tsudaet al., 2009b), while activation of the chemokine receptor CCR2is a key factor involved in post-translational regulation ofP2X4 (Toyomitsu et al., 2012). Fibronectin and IFN-γ canregulate P2X4 expression through the IRF5 transcription factorin microglia (Masuda et al., 2014).

P2X4 activation in spinal microglia leads to the release ofbrain-derived neurotrophic factor (BDNF), which communicatesbetween microglia and spinal interneurons resulting in painhypersensitivity via disinhibition of GABAergic input (Tsudaet al., 2003; Coull et al., 2005; Ulmann et al., 2008; Trang et al.,2009). Allodynia induced through peripheral nerve injury wasreversed by pharmacological blockade of P2X4 receptors in thespinal cord (Tsuda et al., 2003) and P2X4−/− mouse modelshave reduced inflammatory and neuropathic pain (Ulmannet al., 2008). These findings highlight the importance of P2X4in microglial cells in the development of neuropathic pain, asreviewed by Inoue and Tsuda (2012) and Tsuda et al. (2013)and summarized in Figure 1. Furthermore, activated microgliaplay a critical role in the pathogenesis of other diseases suchas spinal cord injury, stroke and neurodegenerative disease (i.e.,Alzheimer’s and Parkinson’s disease), therefore P2X4 may alsocontribute to pathogenesis of these conditions.

Astrocytes are star-shaped glial cells providing a wide varietyof functional support for neurons. P2X1-5 and P2X7 receptorshave been identified at the transcriptional level in situ andin vitro in primary cultured rat cortical astrocytes, hippocampalastrocytes and astrocytes in the nucleus accumbens (Frankeet al., 2001; Fumagalli et al., 2003; Dixon et al., 2004) andprotein expression was further confirmed (Kukley et al., 2001).In rat hippocampus, immunostaining revealed expression ofP2X4 particularly in S100β positive astrocytes in the CA1, CA3and dentate gyrus regions (Kukley et al., 2001). However, in

acutely isolated mouse cortical astrocytes no mRNA expressionfor P2X4 was detected using qRT-PCR. This correlated withelectrophysiology studies showing insensitivity of ATP-inducedcurrents to the positive modulator ivermectin (Jabs et al., 2007).At a functional level, P2X4 responses are not ubiquitously foundsince P2X4-mediated currents were not detected in acutelyisolated cortical astrocytes (Lalo et al., 2008) or hippocampalslices (Jabs et al., 2007). Currently, therefore, there is limitedevidence for P2X4 in astrocytes.

Oligodendrocytes are glial cells primarily responsible formyelination of neurons in the CNS and expression of severaltypes of P2 receptor including P2X4 has been demonstrated inoligodendrocytes and in oligodendrocyte precursor cells (OPCs)(Agresti et al., 2005). Aside from neurons and neuroglial cellsthere is a vast network of blood vessels in the CNS. There is a longstanding interest in the role of P2X4 in peripheral endothelialcells and the regulation of blood vessel tone (Yamamoto et al.,2000, 2006). P2X4 is abundantly expressed in peripheral vesselendothelial cells but it is unclear if this is also true for brainmicrovascular endothelial cells which play an important role inconstitution of the blood brain barrier (BBB) and deteriorationof this barrier is implicated in several neurological disorders.To date expression of P2X4 has been demonstrated in thehCMEC/D3 human microvascular endothelial cell line and themouse microvascular cell line bend.3 (Bintig et al., 2012; Ozakiet al., 2016). Similar to the periphery, brain endothelial P2X4may be involved in regulating shear stress responses in thecerebral vasculature and stimulation of protective factors such asosteopontin to enable ischaemic tolerance (Ozaki et al., 2016).

SUBCELLULAR LOCALISATION OF P2X4

The cellular localisation of most P2X receptors is thought tobe predominantly at the plasma membrane. However, P2X4expression at the cell surface is limited with the majorityof receptors occupying an intracellular residency in restingcells (Robinson and Murrell-Lagnado, 2013). P2X4 containsan additional C-terminal trafficking motif (YxxxGϕ) which

FIGURE 1 | Postulated role of P2X4 signaling in chronic pain. Peripheral nerve injury (PNI) activates microglia in the dorsal horn of the spinal cord. This causesthe upregulation of the P2X4 expression which is modulated by fibronectin and chemokine ligand 21 (CCL21). CCL2 signaling promotes P2X4 trafficking to cellsurface of microglia. Influx of Ca2+ through ATP-stimulated P2X4 activates p38-MAPK and drives the synthesis and SNARE-dependent release of brain-derivedneurotrophic factor (BDNF). After BDNF is released, it acts on its cognate receptor, trkB which consequently downregulates potassium-chloride cotransporter KCC2expression in dorsal horn spinal lamina 1 neurons. This results in increase of intracellular [CI−] and leads to the reduction in anion gradient in dorsal horn which inturn induces depolarization of these neurons. The altered chloride gradient causes GABA to switch its effects from inhibition to excitation. The resultanthyperexcitability in the dorsal horn could underlie the increased sensitivity that is a feature of neuropathic pain.

Frontiers in Pharmacology | www.frontiersin.org 4 May 2017 | Volume 8 | Article 291

fphar-08-00291 May 22, 2017 Time: 17:37 # 5

Stokes et al. P2X4 Function and Pharmacology

facilitates rapid internalization of cell surface P2X4 via AP2and clathrin-dependent endocytosis (Royle et al., 2005). Thesubcellular distribution of P2X4 in many cell types shows apunctate, clustered staining pattern with overlapping markersof intracellular vesicles including recycling endosomes andlysosomes (Bobanovic et al., 2002; Qureshi et al., 2007; Stokesand Surprenant, 2009). Although regulated trafficking is notunique to P2X4 among the P2X receptor family, the abilityof P2X4 to rapidly recycle between the plasma membraneand intracellular compartments is a robust feature studiedby electrophysiological and biochemical methods. There isstrong evidence that P2X4 receptors localize to the plasmamembrane, endosomes, lysosomes, and vacuoles. Insights aboutdynamic P2X4 trafficking were revealed with use of a pH-sensitivecou fluorescent protein, pHluorin. Khakh and colleagueshave recently used P2X4-pHluorin for in vitro trafficking studiesto quantify and track P2X4 distribution within subcellularcompartments (Xu et al., 2014). Similarly, other mechanisticstudies also showed that P2X4 upregulation occurs both viaaltered trafficking and via increased P2X4 gene expression(Toulme et al., 2006, 2010; Raouf et al., 2007; Toulme and Khakh,2012). Recently it was suggested that P2X4 functions as an ATP-gated channel in lysosomes (Huang et al., 2014) and regulatesendolysosomal membrane fusion via Ca2+-dependent activationof calmodulin (Cao et al., 2015).

P2X4 DOWNSTREAM SIGNALINGPATHWAYS

Following ATP activation of P2X4 channels in the plasmamembrane, the rapid influx of cations (Na+ and Ca2+) will causemembrane depolarisation. In addition the significant influx ofCa2+, as has been reported for P2X4, will likely trigger importantdownstream signaling pathways. For example, Ca2+ is importantin neurons in the regulation of neurotransmitter release (Neherand Sakaba, 2008) and in various microglial functions (Farberand Kettenmann, 2006).

Recently, several lines of evidence suggest that P2X4stimulated microglia signal to spinal lamina I dorsal hornneurons causing nociceptive output and that the criticalmicroglia-neuron signaling molecule is BDNF (Coull et al.,2005). Notably, accumulation of BDNF in spinal dorsal hornmicroglia following nerve injury in global P2X4−/− mice showedimpaired ATP-evoked BDNF release which further demonstratedthat P2X4 plays an important role in controlling BDNF releasefrom microglia (Ulmann et al., 2008). The mechanistic gapbetween the activation of P2X4 and release of BDNF frommicroglia was bridged with a study from Salter and colleagues(Trang et al., 2009). Influx of Ca2+ through P2X4 in culturedmicroglia is a critical intracellular step linking stimulationof these receptors to the activation of p38 MAPK. Thisimplicates p38 MAPK activation and BDNF release as a keystep in microglia-neuron communication leading to nerve injuryinduced pain hypersensitivity. However, there may be other celltypes expressing P2X4 involved in pain pathways. Peripheralmacrophages also express P2X4 triggering Ca2+ influx and p38

MAPK phosphorylation (Ulmann et al., 2010). This signalingcould further activate cytosolic PLA2 liberating arachidonic acid(AA) and release of prostaglandin E2 (PGE2). Consequently, therelease of PGE2 leads to hypersensitivity of peripheral nociceptivepathways which is a hallmark of inflammatory pain (Basbaumet al., 2009).

P2X4 IN CNS DISORDERS

As knowledge regarding the expression and the characteristics ofthe individual P2X channels has emerged and expanded sincethey were first cloned in the late 1990s, questions have arisenover the importance of P2X channels in normal physiologicalfunctioning and their functioning in pathological conditions.Information regarding the specific role of P2X4 has been slowerto accumulate largely due to the lack of selective antagonistsfor this P2X subtype (see P2X4 pharmacology section below).The first evidence for a pathological role for P2X4 was inpain processing. A landmark study by Tsuda and colleaguesused antisense P2X4 oligonucleotides and described a majorrole for P2X4 in chronic pain and mechanical allodynia (Tsudaet al., 2003). The development of transgenic mice lacking P2X4expression has since confirmed the role of this ion channel inmodulation of pain signaling (Ulmann et al., 2008, 2010; Tsudaet al., 2009a).

CHRONIC PAIN

Chronic pain is described as persistent pain arising from amodification of pain processing pathways in the spinal cord andcan be labeled as allodynia, hypersensitivity or neuropathic pain(Basbaum et al., 2009; Cohen and Mao, 2014). There are severalreview articles describing purinergic receptors in the context ofpain and it is important to acknowledge here that more thanone purinergic receptor has been implicated in pain – currentlyP2X2/3, P2X7 and P2Y1, P2Y2, P2Y12 are thought to be involvedin the modulation of pain processing (Jarvis, 2010; Tsuda, 2016).

Tsuda and colleagues demonstrated that P2X4 wasupregulated in spinal microglia after nerve injury (ligationof the fifth lumbar spinal nerve) and mediated tactile allodynia(defined as a hypersensitivity to non-painful stimuli) (Tsudaet al., 2003). Blocking P2X4 (with TNP-ATP) or knocking downP2X4 with antisense oligonucleotides reversed the allodyniaphenotype and thus suggested that P2X4 may be a potentialtherapeutic target for this type of neuropathic pain. In 2005it was demonstrated that communication between microgliaand neurons was essential for the development of allodyniaand that the critical factor involved was BDNF (Coull et al.,2005). Stimulation of spinal microglia with ATP inducedBDNF secretion which through activation of TrkB receptorson spinal output neurons, alters Eanion causing disinhibition ofGABAergic inputs from inhibitory spinal neurons. This leadsto hyperexcitability of output neurons in the pain pathway.BDNF was already known to be a pro-nociceptive factor ableto sensitize neurons (Kerr et al., 1999) but this study was the

Frontiers in Pharmacology | www.frontiersin.org 5 May 2017 | Volume 8 | Article 291

fphar-08-00291 May 22, 2017 Time: 17:37 # 6

Stokes et al. P2X4 Function and Pharmacology

first to connect ATP-P2X4-BDNF to the neuronal shift in Eanion.Ulmann et al. (2008) then provided direct evidence for P2X4 inmediating BDNF secretion from spinal microglia using a P2X4-deficient mouse generated by the Rassendren lab. They furtherdemonstrated that P2X4−/− mice did not develop mechanicalhyperalgesia following peripheral nerve injury. This lack ofmechanical hyperalgesia was confirmed using a second globalP2X4 deficient mouse generated by the Ando lab. Behavioraltests demonstrated no difference in acute thermal, mechanicaland chemical induced pain sensing in these P2X4−/− mice butthere was significant suppression of chronic inflammatory pain(induced by CFA injection) and allodynia due to nerve injury(Tsuda et al., 2009a).

More recently, P2X4 has been implicated in toleranceto morphine (Horvath et al., 2010) and morphine-inducedhyperalgesia (Ferrini et al., 2013). Morphine enhanced microglialmigration in vitro in a µ-opioid receptor dependent manner,and antisense P2X4 oligonucleotides reduced the development ofmorphine tolerance in rats (Horvath and DeLeo, 2009; Horvathet al., 2010). Furthermore, Ferrini et al. (2013) demonstratedmorphine hyperalgesia required µ-opioid receptor-mediatedupregulation of P2X4 in spinal microglia. Blocking the BDNF-TrkB pathway reversed the hyperalgesia. Therefore the microglialATP-P2X4-BDNF axis seems to be a central pathway involvedin the setting of pain thresholds. However, recent evidence hasrevealed that this mechanism may be restricted to males sincefemale rodents did not display upregulation of P2X4 on microglia(Sorge et al., 2015; Mapplebeck et al., 2016).

ALCOHOL-RELATED DISORDERS

In the search to identify therapeutic targets for Alcohol UseDisorders (AUD), several studies have demonstrated that P2X4plays a role in alcohol-induced behavior (Yardley et al., 2012;Wyatt et al., 2014). P2X4 are expressed in key brain regionsimplicated in the reinforcing properties of alcohol and otherdrugs (Kimpel et al., 2007). This supports a role for P2X4in alcohol addiction probably via modulation of P2X4 inthe mesolimbic dopamine system, the brain reward system(Khoja et al., 2016). In vitro, P2X4 are inhibited by ethanolconcentrations as low as 5mM and ethanol is thought to blockthe P2X4 channel in the open state but does not affect channeldeactivation (Davies et al., 2005; Ostrovskaya et al., 2011). Crucialresidues that mediates ethanol effects on P2X4 gating are Trp46,His241, Asp331 and Met336 (Xiong et al., 2005; Popova et al.,2010, 2013).

In P2X4−/− mice heightened ethanol intake behavior isobserved over wild-type counterparts suggesting that the P2X4gene may be linked with alcohol intake and/or preference(Asatryan et al., 2011; Franklin et al., 2014). It has been shownthat alcohol-preferring rats have lower expression of the P2X4gene than alcohol-non-preferring rats, suggesting a correlationbetween P2X4 expression and alcohol intake (Kimpel et al., 2007;Franklin et al., 2015). Ivermectin, a broad-spectrum antiparasiticused worldwide in humans and animals, can antagonize theinhibitory action of ethanol on P2X4 and is suggested to interfere

with ethanol binding to the channel (Asatryan et al., 2010). Otheravermectins have been demonstrated to act in a similar fashion(Asatryan et al., 2014; Huynh et al., 2017). Studies also identifya role for P2X4 in regulating dopaminergic neurotransmissionwithin the mesolimbic system. In dopaminergic neurons ofthe mesolimbic system, P2X4 has been shown to play a rolein mediating alcohol-drinking behavior (Franklin et al., 2015).P2X4 receptor activity has also been linked to other behaviorsof associated with dopaminergic neurotransmission, includingmotor control and sensorimotor gating (Khoja et al., 2016).Overall, these findings suggest that alcohol intake may bemodulated by ethanol acting on P2X4 and that pharmacologicalpotentiation of P2X4 by ivermectin or avermectin analogs mayreduce alcohol consumption and preference.

NEUROINFLAMMATORY DISORDERS

Neuroinflammation describes inflammatory changes in the brainand spinal cord associated with activation of the immune system(DiSabato et al., 2016). This is thought to be mediated bymediators such as chemokines, cytokines and second messengers(such as nitric oxide and prostaglandins). Resident immunecells such as microglia are involved, as are endothelial cellsand astrocytes. Microglia regulate cytokines and inflammatoryprocesses in the brain, typically elevated pro-inflammatorycytokines (IL-6, TNF-α, and IL-1β) and chemokines (CCL2,CCL5, CXCL1) define a state of neuroinflammation, as reviewedin DiSabato et al. (2016).

Neurodegenerative diseases such as Alzheimer’s disease andParkinson’s disease are associated with neuroinflammation(Ransohoff, 2016) and microglia appear to be central (Saijo andGlass, 2011; Joers et al., 2016; Wes et al., 2016). P2X4 andthe related purinergic channel P2X7 (Bhattacharya and Biber,2016) are involved in the regulation of microglial pathwaystherefore may also play roles in exacerbating inflammation inthe CNS. P2X4−/− mice (Rassendren mouse model) have anattenuated inflammatory response associated with spinal cordinjury with reduced NLRP1 inflammasome-related signalingpathways (de Rivero Vaccari et al., 2012). P2X4 deficiency alsoreduced the infiltration of peripheral immune cells (neutrophilsand macrophages) to the spinal cord (de Rivero Vaccari et al.,2012).

P2X4 was up-regulated in microglial cells in an animalmodel of multiple sclerosis (autoimmune EAE in rats) (Vazquez-Villoldo et al., 2014). Using LPS as a model of neuroinflammationthe P2X4 antagonists TNP-ATP and 5-BDBD were shown toreduce microglial activation in vivo and reduce the microglialloss in spinal cord. Following i.c.v. injection of LPS, the dentategyrus region of the hippocampus showed classical hallmarks ofmicroglial activation which were blocked by TNP-ATP (Vazquez-Villoldo et al., 2014).

Alcohol abuse is thought to enhance neuroinflammation(Potula et al., 2006) and the P2X4 receptor on microglial cellshas been implicated (Gofman et al., 2014, 2016). In additionneuroinflammatory responses are also associated with ischaemicevents in the brain and studies demonstrate upregulation of P2X4

Frontiers in Pharmacology | www.frontiersin.org 6 May 2017 | Volume 8 | Article 291

fphar-08-00291 May 22, 2017 Time: 17:37 # 7

Stokes et al. P2X4 Function and Pharmacology

in microglia following hypoxia and ischaemia (Wixey et al., 2009;Li et al., 2011). Recently, using a mouse model of middle cerebralartery occlusion (MCAO) endothelial P2X4 was required for theneuroprotective effect of ischaemic preconditioning (transientischaemia followed by reperfusion) (Ozaki et al., 2016). P2X4 isknown to be activated by shear stress generated by conditionsof flow in peripheral endothelial cells (Yamamoto et al., 2000,2006). Mice with a conditional knockout of vascular endothelialP2X4 showed severe deficits after MCAO due to an attenuationin release of the neuroprotective factor osteopontin (Ozaki et al.,2016).

P2X4 PHARMACOLOGY

There has been significant progress in understanding P2Xreceptor pharmacology in recent years. Despite such advances,there remains a paucity of potent and selective pharmacologicaltools for some receptors. Agonists that selectively activate distinctmembers of this family are elusive, and with a few exceptions,progress has been slow when identifying selective inhibitors,some of which are shown in Figure 2. Here we describe what iscurrently known regarding pharmacological modulators of P2X4.

AGONISTS

The most potent agonist of homomeric P2X4 is ATP. Dose-responses for ATP at rat and human P2X4 gave EC50 values of6.9 ± 0.8 and 7.4 ± 0.5 µM, respectively (Soto et al., 1996a;Garcia-Guzman et al., 1997). In the original article characterizingrat P2X4 expressed in Xenopus oocytes the electrophysiologicalprofile of agonists was ATP > 2-methylthio-ATP > CTP > α, β-methylene-ATP > dATP and no response was elicited by ADP,AMP, GTP, adenosine or the β,γ-methylene-ATP analog (Sotoet al., 1996a). For human P2X4 expressed in Xenopus oocytes thesame order of efficacy was demonstrated (Garcia-Guzman et al.,1997). BzATP is also known to act as a partial agonist at rat andhuman P2X4 (Bowler et al., 2003; Stokes et al., 2011).

POSITIVE MODULATORS

One of the most distinguishing features of P2X4 is potentiationby ivermectin – a macrocyclic lactone derived from the selectivehydrogenation of avermectin B1, a natural product synthesizedby Streptomyces avermitilis. Ivermectin is a mixture of 22,23-dihydroavermectin B1a and B1b in an approximate 80:20 ratio(Laing et al., 2017). Use of ivermectin as a successful anti-helminthic drug is due to its positive allosteric modulationof glutamate–gated chloride channels in nematode worms.Ivermectin also potentiates responses at vertebrate GABA(A) andnicotinic α7 receptors (Krušek and Zemková, 1994; Krause et al.,1998). Of all purinergic receptors, P2X4 is the most sensitive toivermectin (Khakh et al., 1999b) although a recent report claimssome potentiation of human P2X7 responses by ivermectin(Nörenberg et al., 2012). Extracellular, but not intracellular,application of ivermectin (≤10 µM) potentiates P2X4 currents

and delays channel deactivation (Khakh et al., 1999b; Priel andSilberberg, 2004). These observations are likely the result ofivermectin binding to separate sites on P2X4, a high affinityivermectin binding site (pEC50 6.6) which increases maximalcurrent activated by saturating concentrations of ATP, and alow affinity site (pEC50 5.7) which slows channel deactivation(Priel and Silberberg, 2004). Ivermectin appears to interact withthe transmembrane domains of P2X4 and lateral fenestrations(Jelinkova et al., 2006; Silberberg et al., 2007; Hattori and Gouaux,2012; Samways et al., 2012; Rokic et al., 2013) including a bindingpocket which overlaps with an inhibitory ethanol binding site(Asatryan et al., 2010).

Avermectin analogs in addition to ivermectin also havepositive modulator activity at P2X4. These include avermectin,emamectin, doramectin (Silberberg et al., 2007) and morerecently abamectin, selamectin, and moxidectin (Asatryan et al.,2014; Huynh et al., 2017). Of these avermectin, abamectin andmoxidectin have similar effects to ivermectin in preventing theaction of ethanol on P2X4, but have less effect on GABA(A)receptors (Asatryan et al., 2014; Huynh et al., 2017).

There is also evidence for positive allosteric modulation ofP2X4 by cibacron blue, an isomer of reactive blue-2, in functionalstudies. Potentiation of P2X4 responses were attributable to anincrease in the apparent affinity of ATP for the rat P2X4 (Milleret al., 1998). This potentiation of P2X4 responses is smaller thanthat of ivermectin and was not demonstrated in cells expressinghuman P2X4 (Garcia-Guzman et al., 1997).

BROAD SPECTRUM PURINERGICRECEPTOR ANTAGONISTS

P2X4 receptors are generally less sensitive to the broadP2X antagonists, such as suramin and pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS). Suramin shows veryweak activity at mouse, rat and human P2X4 orthologs withmaximal inhibition of P2X4 currents ranging between 11 and35% with pIC50 > 4 for all orthologs (Jones et al., 2000). Incontrast, PPADS fully inhibits human and mouse P2X4 (pIC50∼ 5 for both), whilst the rat receptor is relatively insensitiveto PPADS (Jones et al., 2000). It has been hypothesized thatPPADS acts, in part, by forming a Schiff base with a lysineresidue in P2X1 and P2X2. However, this residue in the P2X4is replaced by a glutamate at the analogous position (Glu249)and when it is replaced by a lysine, the resultant P2X4 mutantis sensitive to inhibition by PPADS. Notably, the human P2X4has only one lysine (Lys127), which is absent in the rat P2X4,conferring sensitivity to PPADS, and mutation of the residue toa lysine in the rat P2X4 (Asn127Lys) did not produce a PPADS-sensitive channel (Buell et al., 1996). Critically, the increasedsensitivity of the human P2X4 to PPADS inhibition might notbe only due to the different ability of PPADS to form Schiff basevia lysine residues. In addition, several studies have confirmedthat 2′,3′-O-(2,4,6-trinitrophenyl) ATP (TNP-ATP), which is apotent antagonist at P2X1, P2X3 and P2X2/3 receptors, acts asa weakly effective antagonist (IC50 15.2 µM) at P2X4 receptors(Virginio et al., 1998). TNP-ATP is a competitive antagonist at

Frontiers in Pharmacology | www.frontiersin.org 7 May 2017 | Volume 8 | Article 291

fphar-08-00291 May 22, 2017 Time: 17:37 # 8

Stokes et al. P2X4 Function and Pharmacology

FIGURE 2 | Chemical structures of P2X4 antagonists.

P2X4 and can displace [35S]ATPγ S binding to human P2X4(Hernandez-Olmos et al., 2012; Abdelrahman et al., 2016). Thepotency of TNP-ATP at P2X4 estimated by Ca2+ influx assaysin 1321N1 astrocytoma cells was 1.46–4.22 µM dependent onspecies (Abdelrahman et al., 2016).

DIVALENT CATIONS AND pH

With regard to ions, P2X4 is amongst the most sensitive P2receptor to potentiation by zinc ions (Zn2+). Low concentrations(1–5 µM) of extracellular Zn2+ increases the gating efficiencyof human or rat P2X4 without affecting the maximal response,however, high (>100 µM) extracellular Zn2+ inhibits P2X4currents in a voltage-dependent fashion (Garcia-Guzman et al.,1997; Wildman et al., 1999; Acuna-Castillo et al., 2000). Similarly,the potentiating effects of Zn2+ are mimicked by Cd2+, however,Cu2+ modifies P2X4 activity in the opposite direction causinginhibition (Acuna-Castillo et al., 2000; Coddou et al., 2005).High concentrations of extracellular Cu2+ (300 µM) inhibit ratP2X4 in a non-competitive and voltage-independent fashion andthis effect can be seen with other metal cations such as Hg2+

(Coddou et al., 2005). His140 plays a crucial role in the inhibitoryaction of Cu2+ but this was not required for the action of Zn2+

at P2X4 (Coddou et al., 2003). Moreover, Asp138 was furtheridentified as important for Cu2+ binding at P2X4 and Cys132was critical for Zn2+ potentiation at P2X4 (Coddou et al., 2007).

These residues were validated by an NMR resolved structure ofrat P2X4 extracellular domain as key residues that coordinateCu2+ binding (Igawa et al., 2015).

The activity of P2X4 is not only regulated by metals andions, but is also modulated by extracellular H+, such thatpH < 6 completely inhibits channel activity and responses arepotentiated above physiological pH (Wildman et al., 1999; Clarkeet al., 2000). One key residue, His286, has been identified inconveying pH-sensitivity to P2X4 (Clarke et al., 2000). Inhibitionby low pH may be an important physiological regulator ofP2X4 receptor since P2X4 displays a predominant lysosomaldistribution (Qureshi et al., 2007) where it appears to functionas a lysosomal ion channel in addition to its plasma membranerole (Huang et al., 2014). In lysosomes the luminal pH is typicallyacidic with a resting pH 4.6 maintained by vacuolar H+-ATPases.Under these conditions P2X4 was not active but followingalkalinisation of lysosomes with NH4Cl, ATP-induced responsescould be recorded (Huang et al., 2014).

ANTIDEPRESSANTS

Recent studies have documented that some antidepressantsmay inhibit P2X4 receptors, however, this evidence remainscontroversial in view of contrasting findings. P2X4 is inhibitedby several clinically used antidepressants including paroxetine,fluoxetine, desipramine, fluvoxamine, nortriptyline, and

Frontiers in Pharmacology | www.frontiersin.org 8 May 2017 | Volume 8 | Article 291

fphar-08-00291 May 22, 2017 Time: 17:37 # 9

Stokes et al. P2X4 Function and Pharmacology

TABLE 1 | P2X4 modulators and antagonists.

Compound Nature Potency at P2X4 IC50 µM (species) Selectivity Reference

5-BDBD Competitive 1.6 (Human) Not reported Balazs et al., 2013;Abdelrahman et al., 2016

PSB-12054 Allosteric 0.189 (Human) 30-fold (P2X1) Hernandez-Olmos et al., 2012

2.10 (Rat) 50-fold (P2X2, 3, 7)

1.77 (Mouse)

PSB-12062 Allosteric 1.38 (Human) 35-fold (P2X1-3, 7) Hernandez-Olmos et al., 2012

0.928 (Rat)

1.76 (Mouse)

BX-430 Non-competitive allosteric 0.54 (Human) 10-fold (P2X1-3, 5) Ase et al., 2015

100-fold (P2X7)

1.89 (Zebrafish)

Carbamazepine derivative Negative allosteric modulator 3.44 (Human) 2-fold (P2X1, 3) Tian et al., 2014

30-fold (P2X2, 7)

54.6 (Rat)

14.9 (Mouse)

NP-1815-PX Not reported 0.26 (Human) Not reported Matsumura et al., 2016

Ivermectin Positive allosteric modulator 0.25 (Human) Acts on human P2X7 Priel and Silberberg, 2004;Nörenberg et al., 2012

Cibacron blue Potentiator Not determined Not reported Miller et al., 1998

clomipramine (Nagata et al., 2009). Of these paroxetine isthe most potent, non-competitively inhibiting human P2X4heterologously expressed in 1321N1 cells at a pIC50 of 5.7(Nagata et al., 2009; Abdelrahman et al., 2016). The studiessuggest that such compounds may mediate analgesic effects inneuropathic pain models via the inhibition of P2X4 (Nagataet al., 2009; Zarei et al., 2014; Yamashita et al., 2016). However,paroxetine is much more potent as a serotonin reuptakeinhibitor than an inhibitor of P2X4. There is also evidence thatantidepressants may affect lysosomal trafficking of P2X4 incerebellar microglial cells (Toulme et al., 2010). Furthermore,the hypothesis that the widely used tricyclic antidepressantamitriptyline may exert analgesic effects through P2X4 inhibitionhas also been tested (Sim and North, 2010). Amitriptyline appliedacutely at 10 µM or by pre-incubation for several hours, had noeffect on human P2X4 channel activity though modest inhibitoryeffects of 10 µM amitriptyline were observed at mouse and ratP2X4 (Sim and North, 2010). It is therefore highly unlikely thatamitriptyline mediates analgesic effects via P2X4 inhibition inhuman subjects.

STATINS AND CHOLESTEROLDEPLETING AGENTS

Statins have been described as the most effective class of drugsto reduce serum cholesterol levels (Oesterle et al., 2017). TheHMG-CoA reductase inhibitor fluvastatin inhibited the activityof heterologously expressed human P2X4 and native P2X4 inhuman monocytes (Li and Fountain, 2012). The activity offluvastatin is likely to be due to depletion of cellular cholesterolas its inhibitory action was mimicked by methyl-β-cyclodextrinand filipin III (Li and Fountain, 2012). Some further studies

have shown association of P2X4 within lipid rafts (Allsopp et al.,2010).

RECENT ADVANCES IN P2X4PHARMACOLOGICAL AGENTS

Research surrounding P2X4 has been greatly hindered due to thelack of selective receptor antagonists. For a long time, researchershad to rely on non-selective P2X receptor antagonists suchas TNP-ATP, BBG and paroxetine to study P2X4. However,these agents possess low affinity for P2X4 and are significantlymore potent at other targets. Recently growing evidence thathighlights the importance of P2X4 in health and disease hastriggered interest in the development of selective receptorantagonists (Table 1). The discovery of selective P2X4 antagonistswill, therefore, present a new avenue for scientific research toselectively target P2X4 therapeutically.

5-BDBD

The benzodiazepine derivative 5-BDBD (5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one) was oneof the first selective P2X4 antagonists to be described in theliterature (Balazs et al., 2013). The original experimental detailsare documented in a patent (Fischer et al., 2005), however,5-BDBD is now commercially available. This compound has beenreported as a moderately potent and selective P2X4 antagonistwith an IC50 value of 1.2 µM in HEK-293 cells expressinghuman P2X4 (Balazs et al., 2013). The application of twodifferent concentrations of 5-BDBD resulted in a rightward shiftin ATP dose-response curve indicating that 5-BDBD acts as acompetitive antagonist to P2X4 (Balazs et al., 2013). However,

Frontiers in Pharmacology | www.frontiersin.org 9 May 2017 | Volume 8 | Article 291

fphar-08-00291 May 22, 2017 Time: 17:37 # 10

Stokes et al. P2X4 Function and Pharmacology

radioligand binding assays illustrated that 5-BDBD was unableto displace [35S]ATPγS binding to P2X4 indicating that ratherthan acting as a true competitive antagonist, it has an allostericmechanism at P2X4 (Abdelrahman et al., 2016).

N-SUBSTITUTED PHENOXAZINEDERIVATIVES

More recently, compounds derived from N-substitutedphenoxazine were identified as potent and selective allostericP2X4 antagonists (Hernandez-Olmos et al., 2012). One of thederivatives, N-(Benzyloxycarbonyl) phenoxazine (PSB-12054), isan extremely potent antagonist of the human P2X4 (IC50 valueof 0.189 µM), but is less potent toward rat P2X4 (IC50 valueof 2.1 µM) and mouse P2X4 (IC50 value of 1.77 µM). It hasbeen reported to possess high selectivity for human P2X4 withover 50-fold against P2X2, P2X3 and P2X7 and over 30-foldagainst P2X1 (Hernandez-Olmos et al., 2012). The second lesspotent, but more water-soluble, of the two derivatives is N-(p-methylphenylsulfonyl)phenoxazine (PSB-12062). PSB-12062 wasshown to have similar potency in all three species: human P2X4(IC50 value of 1.38 µM), rat P2X4 (IC50 value of 0.928 µM)and mouse P2X4 (IC50 value of 1.76 µM) (Hernandez-Olmoset al., 2012). PSB-12062 has been shown to be allosteric in naturewith a 35-fold selectivity toward P2X4 versus P2X1, P2X2, P2X3,and P2X7. However, PSB-12062 was unable to completely blockATP-induced P2X4-mediated calcium influx even when used athigh concentrations (>30 µM) (Hernandez-Olmos et al., 2012).A third compound also derived from N-substituted phenoxazine,PSB-12253, was used in a study on HUVECs to investigate P2X4responses. The effects of this selective antagonist were confirmedby siRNA in HUVECs (Sathanoori et al., 2015).

CARBAMAZEPINE DERIVATIVES

Several carbamazepine derivatives were investigated as potentialP2X4 antagonists, with N,N-diisopropyl-5H-dibenz[b,f]azepine-5-carboxamide being found to be the most potent toward humanP2X4 (IC50 of 3.44 µM) although less potent at mouse and ratP2X4 (Tian et al., 2014). Despite its potency toward human P2X4,it appeared to lack selectivity against two other P2X subtype(P2X1 and P2X3) but was selective versus P2X2 and P2X7.Further optimization will be required to improve the selectivityof this compound.

BX430

One of the most recently discovered P2X4 antagonists isthe phenylurea BX430 (1-(2,6-dibromo-4-isopropyl-phenyl)-3-(3-pyriudyl)urea which has been shown through a patch-clamp

study to have sub-micromolar potency (IC50 value of 0.54 µM)and a 10 – 100 fold selectivity toward P2X4 versus other P2Xsubtypes (Ase et al., 2015). Acting through a non-competitiveallosteric route, BX430 has been shown to be able to potentlyantagonize zebrafish P2X4 but has no effect on rat and mouseP2X4 orthologs (Ase et al., 2015). BX430 was seen to completelyabolish ivermectin facilitated pore formation and, according tothe authors, BX430 is the only known compound to inhibit P2X4mediated pore formation (Ase et al., 2015).

NP-1815-PX

Screening a chemical library identified the compound NP-1815-PX (5-[3-(5-thioxo-4H-[1,2,4]oxadiazol-3-yl)phenyl]-1H-naphtho[1,2-b][1,4]diazepine-2,4(3H,5H)-dione) as a novelhuman P2X4 antagonist (IC50 value of 0.26 µM) (Matsumuraet al., 2016). The inhibitory effect of NP-1815-PX was alsoshown on rat and mouse P2X4 and it showed selectivity againstother P2X receptors (Matsumura et al., 2016). Intrathecaladministration of NP-1815-PX alleviated nerve damage-associated mechanical allodynia in a chronic pain model andin a herpetic pain model without altering acute pain sensing(Matsumura et al., 2016).

FUTURE PERSPECTIVES

The discovery of new pharmacological tools, some of which arenow available commercially, will facilitate our understanding ofP2X4 cellular function in health and disease. In addition, suchtools may provide chemical scaffolds for the development of newdrugs that may be beneficial in the treatment of neuropathicpain. P2X4-selective antagonists with high potency, no species-restricted effect, water-solubility and, most importantly, an anti-allodynia effect in rodent chronic pain models remain to beidentified.

AUTHOR CONTRIBUTIONS

LS initiated, designed, co-wrote, and edited the article. JL co-wrote the article and prepared the table. LB co-wrote andprepared the Figures. KD co-wrote the article. SF co-wrote andedited the article.

FUNDING

Both LS and SF are funded by BBSRC.

Frontiers in Pharmacology | www.frontiersin.org 10 May 2017 | Volume 8 | Article 291

fphar-08-00291 May 22, 2017 Time: 17:37 # 11

Stokes et al. P2X4 Function and Pharmacology

REFERENCESAbdelrahman, A., Namasivayam, V., Hinz, S., Schiedel, A. C., Köse, M., Burton, M.,

et al. (2016). Characterization of P2X4 receptor agonists and antagonists bycalcium influx and radioligand binding studies. Biochem. Pharmacol. 125,41–54. doi: 10.1016/j.bcp.2016.11.016

Acuna-Castillo, C., Morales, B., and Huidobro-Toro, J. P. (2000). Zinc andcopper modulate differentially the P2X4 receptor. J. Neurochem. 74, 1529–1537.doi: 10.1046/j.1471-4159.2000.0741529.x

Agresti, C., Meomartini, M. E., Amadio, S., Ambrosini, E., Volonté, C.,Aloisi, F., et al. (2005). ATP regulates oligodendrocyte progenitor migration,proliferation, and differentiation: involvement of metabotropic P2 receptors.Brain Res. Rev. 48, 157–165. doi: 10.1016/j.brainresrev.2004.12.005

Allsopp, R. C., Lalo, U., and Evans, R. J. (2010). Lipid raft association andcholesterol sensitivity of p2x1-4 receptors for atp: chimeras and point mutantsidentify intracellular amino-terminal residues involved in lipid regulation ofp2x1 receptors. J. Biol. Chem. 285, 32770–32777. doi: 10.1074/jbc.M110.148940

Amadio, S., Montilli, C., Picconi, B., Calabresi, P., and Volonte, C. (2007).Mapping P2X and P2Y receptor proteins in striatum and substantia nigra: animmunohistological study. Purinergic Signal. 3, 389–398. doi: 10.1007/s11302-007-9069-8

Antonio, L. S., Stewart, A. P., Xu, X. J., Varanda, W. A., Murrell-Lagnado, R. D.,and Edwardson, J. M. (2011). P2X4 receptors interact with both P2X2 andP2X7 receptors in the form of homotrimers. Br. J. Pharmacol. 163, 1069–1077.doi: 10.1111/j.1476-5381.2011.01303.x

Asatryan, L., Nam, H. W., Lee, M. R., Thakkar, M. M., Saeed Dar, M., Davies,D. L., et al. (2011). Implication of the purinergic system in alcohol use disorders.Alcohol. Clin. Exp. Res. 35, 584–594. doi: 10.1111/j.1530-0277.2010.01379.x

Asatryan, L., Popova, M., Perkins, D., Trudell, J. R., Alkana, R. L., and Davies, D. L.(2010). Ivermectin antagonizes ethanol inhibition in purinergic P2X4 receptors.J. Pharmacol. Exp. Ther. 334, 720–728. doi: 10.1124/jpet.110.167908

Asatryan, L., Yardley, M. M., Khoja, S., Trudell, J. R., Hyunh, N., Louie, S. G., et al.(2014). Avermectins differentially affect ethanol intake and receptor function:implications for developing new therapeutics for alcohol use disorders. Int. J.Neuropsychopharmacol. 17, 907–916. doi: 10.1017/S1461145713001703

Ase, A. R., Honson, N. S., Zaghdane, H., Pfeifer, T. A., and Seguela, P. (2015).Identification and characterization of a selective allosteric antagonist of humanP2X4 receptor channels. Mol. Pharmacol. 87, 606–616. doi: 10.1124/mol.114.096222

Balazs, B., Danko, T., Kovacs, G., Koles, L., Hediger, M. A., and Zsembery, A.(2013). Investigation of the inhibitory effects of the benzodiazepine derivative,5-BDBD on P2X4 purinergic receptors by two complementary methods. CellPhysiol. Biochem. 32, 11–24. doi: 10.1159/000350119

Banke, T. G., Chaplan, S. R., and Wickenden, A. D. (2010). Dynamic changes in theTRPA1 selectivity filter lead to progressive but reversible pore dilation. Am. J.Physiol. Cell Physiol. 298, C1457–C1468. doi: 10.1152/ajpcell.00489.2009

Bardoni, R., Goldstein, P. A., Lee, C. J., Gu, J. G., and Macdermott, A. B. (1997).ATP P2X Receptors Mediate fast synaptic transmission in the dorsal horn ofthe rat spinal cord. J. Neurosci. 17, 5297–5304.

Basbaum, A. I., Bautista, D. M., Scherrer, G., and Julius, D. (2009). Cellular andmolecular mechanisms of pain. Cell 139, 267–284. doi: 10.1016/j.cell.2009.09.028

Baxter, A. W., Choi, S. J., Sim, J. A., and North, R. A. (2011). Role of P2X4 receptorsin synaptic strengthening in mouse CA1 hippocampal neurons. Eur. J. Neurosci.34, 213–220. doi: 10.1111/j.1460-9568.2011.07763.x

Bernier, L. P., Ase, A. R., Boue-Grabot, E., and Seguela, P. (2012). P2X4 receptorchannels form large noncytolytic pores in resting and activated microglia. Glia60, 728–737. doi: 10.1002/glia.22301

Bhattacharya, A., and Biber, K. (2016). The microglial ATP-gated ion channel P2X7as a CNS drug target. Glia 64, 1772–1787. doi: 10.1002/glia.23001

Biber, K., Tsuda, M., Tozaki-Saitoh, H., Tsukamoto, K., Toyomitsu, E., Masuda, T.,et al. (2011). Neuronal CCL21 up-regulates microglia P2X4 expression andinitiates neuropathic pain development. EMBO J. 30, 1864–1873. doi: 10.1038/emboj.2011.89

Bintig, W., Begandt, D., Schlingmann, B., Gerhard, L., Pangalos, M., Dreyer, L.,et al. (2012). Purine receptors and Ca(2+) signalling in the human blood-brain barrier endothelial cell line hCMEC/D3. Purinergic Sig. 8, 71–80.doi: 10.1007/s11302-011-9262-7

Bo, X., Kim, M., Nori, S. L., Schoepfer, R., Burnstock, G., and North, R. A. (2003).Tissue distribution of P2X4 receptors studied with an ectodomain antibody.CellTissue Res. 313, 159–165. doi: 10.1007/s00441-003-0758-5

Bobanovic, L. K., Royle, S. J., and Murrell-Lagnado, R. D. (2002). P2X receptortrafficking in neurons is subunit specific. J. Neurosci. 22, 4814–4824.

Boumechache, M., Masin, M., Edwardson, J. M., Gorecki, D. C., and Murrell-Lagnado, R. (2009). Analysis of assembly and trafficking of native P2X4 andP2X7 receptor complexes in rodent immune cells. J. Biol. Chem. 284, 13446–13454. doi: 10.1074/jbc.M901255200

Bowler, J. W., Jayne Bailey, R., Alan North, R., and Surprenant, A. (2003). P2X4,P2Y1 and P2Y2 receptors on rat alveolar macrophages. Br. J. Pharmacol. 140,567–575. doi: 10.1038/sj.bjp.0705459

Browne, L. E., Compan, V., Bragg, L., and North, R. A. (2013). P2X7 receptorchannels allow direct permeation of nanometer-sized dyes. J. Neurosci. 33,3557–3566. doi: 10.1523/JNEUROSCI.2235-12.2013

Buell, G., Lewis, C., Collo, G., North, R. A., and Surprenant, A. (1996). Anantagonist-insensitive P2X receptor expressed in epithelia and brain. EMBO J.15, 55–62.

Burnstock, G. (2006). Historical review: ATP as a neurotransmitter. TrendsPharmacol. Sci. 27, 166–176. doi: 10.1016/j.tips.2006.01.005

Burnstock, G., and Kennedy, C. (2011). “P2X Receptors in Health and Disease,” inAdvances in Pharmacology, Chap. 11, eds A. J. Kenneth and L. Joel (Cambridge,MA: Academic Press), 333–372. doi: 10.1016/b978-0-12-385526-8.00011-4

Cao, Q., Zhong, X. Z., Zou, Y., Murrell-Lagnado, R., Zhu, M. X., and Dong,X. P. (2015). Calcium release through P2X4 activates calmodulin to promoteendolysosomal membrane fusion. J. Cell Biol. 209, 879–894. doi: 10.1083/jcb.201409071

Cardoso, A. M., Schetinger, M. R., Correia-De-Sa, P., and Sevigny, J. (2015). Impactof ectonucleotidases in autonomic nervous functions. Auton. Neurosci. 191,25–38. doi: 10.1016/j.autneu.2015.04.014

Cham, J. L., Owens, N. C., Barden, J. A., Lawrence, A. J., and Badoer, E. (2006).P2X purinoceptor subtypes on paraventricular nucleus neurones projectingto the rostral ventrolateral medulla in the rat. Exp. Physiol. 91, 403–411.doi: 10.1113/expphysiol.2005.032409

Chung, M. K., Guler, A. D., and Caterina, M. J. (2008). TRPV1 shows dynamicionic selectivity during agonist stimulation. Nat. Neurosci. 11, 555–564.doi: 10.1038/nn.2102

Clarke, C. E., Benham, C. D., Bridges, A., George, A. R., and Meadows, H. J.(2000). Mutation of histidine 286 of the human P2X4 purinoceptor removesextracellular pH sensitivity. J. Physiol. 523(Pt 3), 697–703. doi: 10.1111/j.1469-7793.2000.00697.x

Coddou, C., Acuna-Castillo, C., Bull, P., and Huidobro-Toro, J. P. (2007).Dissecting the facilitator and inhibitor allosteric metal sites of the P2X4 receptorchannel: critical roles of CYS132 for zinc potentiation and ASP138 for copperinhibition. J. Biol. Chem. 282, 36879–36886. doi: 10.1074/jbc.M706925200

Coddou, C., Lorca, R. A., Acuna-Castillo, C., Grauso, M., Rassendren, F., andHuidobro-Toro, J. P. (2005). Heavy metals modulate the activity of thepurinergic P2X4 receptor.Toxicol. Appl. Pharmacol. 202, 121–131. doi: 10.1016/j.taap.2004.06.015

Coddou, C., Morales, B., Gonzalez, J., Grauso, M., Gordillo, F., Bull, P., et al.(2003). Histidine 140 plays a key role in the inhibitory modulation of the P2X4nucleotide receptor by copper but not zinc. J. Biol. Chem. 278, 36777–36785.doi: 10.1074/jbc.M305177200

Cohen, S. P., and Mao, J. (2014). Neuropathic pain: mechanisms and their clinicalimplications. BMJ 348:f7656. doi: 10.1136/bmj.f7656

Coull, J. A. M., Beggs, S., Boudreau, D., Boivin, D., Tsuda, M., Inoue, K., et al.(2005). BDNF from microglia causes the shift in neuronal anion gradientunderlying neuropathic pain. Nature 438, 1017–1021. doi: 10.1038/nature04223

Davies, D. L., Kochegarov, A. A., Kuo, S. T., Kulkarni, A. A., Woodward, J. J.,King, B. F., et al. (2005). Ethanol differentially affects ATP-gated P2X3 andP2X4 receptor subtypes expressed in Xenopus oocytes. Neuropharmacology 49,243–253. doi: 10.1016/j.neuropharm.2005.03.015

de Rivero Vaccari, J. P., Bastien, D., Yurcisin, G., Pineau, I., Dietrich, W. D., DeKoninck, Y., et al. (2012). P2X4 receptors influence inflammasome activationafter spinal cord injury. J. Neurosci. 32, 3058–3066. doi: 10.1523/JNEUROSCI.4930-11.2012

Di Virgilio, F., Evans, R. J., Jarvis, M. F., Kennedy, C., Khakh, B. S.,Pellegatti, P., et al. (2015). P2X receptors; P2X4. IUPHAR/BPS Guide

Frontiers in Pharmacology | www.frontiersin.org 11 May 2017 | Volume 8 | Article 291

fphar-08-00291 May 22, 2017 Time: 17:37 # 12

Stokes et al. P2X4 Function and Pharmacology

to Pharmacology. Available at: http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=77.

DiSabato, D. J., Quan, N., and Godbout, J. P. (2016). Neuroinflammation: the devilis in the details. J. Neurochem. 139, 136–153. doi: 10.1111/jnc.13607

Dixon, S. J., Yu, R., Panupinthu, N., and Wilson, J. X. (2004). Activation of P2nucleotide receptors stimulates acid efflux from astrocytes. Glia 47, 367–376.doi: 10.1002/glia.20048

Egan, T. M., and Khakh, B. S. (2004). Contribution of calcium ions to P2X channelresponses. J. Neurosci. 24, 3413–3423. doi: 10.1523/JNEUROSCI.5429-03.2004

Farber, K., and Kettenmann, H. (2006). Functional role of calcium signals formicroglial function. Glia 54, 656–665. doi: 10.1002/glia.20412

Ferrini, F., Trang, T., Mattioli, T.-A. M., Laffray, S., Del’guidice, T., Lorenzo,L.-E., et al. (2013). Morphine hyperalgesia gated through microglia-mediated disruption of neuronal Cl- homeostasis. Nat. Neurosci 16, 183–192.doi: 10.1038/nn.3295

Fischer, R., Kalthof, B., Gruetzmann, R., Woltering, E., Stelte-Ludwig, B., andWuttke, M. (2005). Benzofuro-1,4-diazepin-2-one Derivatives. US CA 2519987A1.

Franke, H., Grosche, J., Schädlich, H., Krügel, U., Allgaier, C., and Illes, P. (2001).P2X receptor expression on astrocytes in the nucleus accumbens of rats.Neuroscience 108, 421–429. doi: 10.1016/S0306-4522(01)00416-X

Franklin, K. M., Asatryan, L., Jakowec, M. W., Trudell, J. R., Bell, R. L., andDavies, D. L. (2014). P2X4 receptors (P2X4Rs) represent a novel target forthe development of drugs to prevent and/or treat alcohol use disorders. Front.Neurosci. 8:176. doi: 10.3389/fnins.2014.00176

Franklin, K. M., Hauser, S. R., Lasek, A. W., Bell, R. L., and Mcbride, W. J. (2015).Involvement of purinergic P2X4 receptors in alcohol intake of high-alcohol-drinking (HAD) rats. Alcohol. Clin. Exp. Res. 39, 2022–2031. doi: 10.1111/acer.12836

Fumagalli, M., Brambilla, R., D’ambrosi, N., Volonté, C., Matteoli, M., Verderio, C.,et al. (2003). Nucleotide-mediated calcium signaling in rat cortical astrocytes:Role of P2X and P2Y receptors. Glia 43, 218–230. doi: 10.1002/glia.10248

Gao, C., Yu, Q., Xu, H., Zhang, L., Liu, J., Jie, Y., et al. (2015). Roles of thelateral fenestration residues of the P2X(4) receptor that contribute to thechannel function and the deactivation effect of ivermectin. Purinergic Signal.11, 229–238. doi: 10.1007/s11302-015-9448-5

Garcia-Guzman, M., Soto, F., Gomez-Hernandez, J. M., Lund, P. E., andStuhmer, W. (1997). Characterization of recombinant human P2X4 receptorreveals pharmacological differences to the rat homologue. Mol. Pharmacol. 51,109–118.

Gofman, L., Cenna, J. M., and Potula, R. (2014). P2X4 receptor regulates alcohol-induced responses in microglia. J. Neuroimmune Pharmacol. 9, 668–678.doi: 10.1007/s11481-014-9559-8

Gofman, L., Fernandes, N. C., and Potula, R. (2016). Relative role of Akt, ERKand CREB in alcohol-induced microglia P2X4R Receptor Expression. AlcoholAlcohol. 51, 647–654. doi: 10.1093/alcalc/agw009

Guo, L. H., Trautmann, K., and Schluesener, H. J. (2005). Expression of P2X4receptor by lesional activated microglia during formalin-induced inflammatorypain. J. Neuroimmunol. 163, 120–127. doi: 10.1016/j.jneuroim.2005.03.007

Hattori, M., and Gouaux, E. (2012). Molecular mechanism of ATP binding andion channel activation in P2X receptors. Nature 485, 207–212. doi: 10.1038/nature11010

He, M.-L., Gonzalez-Iglesias, A. E., and Stojilkovic, S. S. (2003). Role of NucleotideP2 receptors in calcium signaling and prolactin release in pituitary lactotrophs.J. Biol. Chem. 278, 46270–46277. doi: 10.1074/jbc.M309005200

Hernandez-Olmos, V., Abdelrahman, A., El-Tayeb, A., Freudendahl, D.,Weinhausen, S., and Muller, C. E. (2012). N-substituted phenoxazine andacridone derivatives: structure-activity relationships of potent P2X4 receptorantagonists. J. Med. Chem. 55, 9576–9588. doi: 10.1021/jm300845v

Horvath, R. J., and DeLeo, J. A. (2009). Morphine enhances microglial migrationthrough modulation of P2X4 receptor signaling. J. Neurosci. 29, 998–1005.doi: 10.1523/JNEUROSCI.4595-08.2009

Horvath, R. J., Romero-Sandoval, E. A., and De Leo, J. A. (2010). Inhibition ofmicroglial P2X4 receptors attenuates morphine tolerance, Iba1, GFAP and muopioid receptor protein expression while enhancing perivascular microglialED2. Pain 150, 401–413. doi: 10.1016/j.pain.2010.02.042

Huang, P., Zou, Y., Zhong, X. Z., Cao, Q., Zhao, K., Zhu, M. X., et al. (2014).P2X4 forms functional ATP-activated cation channels on lysosomal membranes

regulated by luminal pH. J. Biol. Chem. 289, 17658–17667. doi: 10.1074/jbc.M114.552158

Huynh, N., Arabian, N., Naito, A., Louie, S., Jakowec, M. W., Asatryan, L., et al.(2017). Preclinical development of moxidectin as a novel therapeutic for alcoholuse disorder. Neuropharmacology 113, 60–70. doi: 10.1016/j.neuropharm.2016.09.016

Idzko, M., Ferrari, D., and Eltzschig, H. K. (2014). Nucleotide signalling duringinflammation. Nature 509, 310–317. doi: 10.1038/nature13085

Igawa, T., Abe, Y., Tsuda, M., Inoue, K., and Ueda, T. (2015). Solution structure ofthe rat P2X4 receptor head domain involved in inhibitory metal binding. FEBSLett. 589, 680–686. doi: 10.1016/j.febslet.2015.01.034

Inoue, K., and Tsuda, M. (2012). P2X4 receptors of microglia in neuropathicpain. CNS Neurol. Disord. Drug Targets 11, 699–704. doi: 10.2174/187152712803581065

Ito, K., Chihara, Y., Iwasaki, S., Komuta, Y., Sugasawa, M., and Sahara, Y. (2010).Functional ligand-gated purinergic receptors (P2X) in rat vestibular ganglionneurons. Hear. Res. 267, 89–95. doi: 10.1016/j.heares.2010.03.081

Jabs, R., Matthias, K., Grote, A., Grauer, M., Seifert, G., and Steinhauser, C. (2007).Lack of P2X receptor mediated currents in astrocytes and GluR type glial cellsof the hippocampal CA1 region. Glia 55, 1648–1655. doi: 10.1002/glia.20580

Jarvis, M. F. (2010). The neural-glial purinergic receptor ensemble in chronic painstates. Trends Neurosci. 33, 48–57. doi: 10.1016/j.tins.2009.10.003

Jelinkova, I., Yan, Z., Liang, Z., Moonat, S., Teisinger, J., Stojilkovic, S. S., et al.(2006). Identification of P2X4 receptor-specific residues contributing to theivermectin effects on channel deactivation. Biochem. Biophys. Res. Commun.349, 619–625. doi: 10.1016/j.bbrc.2006.08.084

Jo, Y. H., Donier, E., Martinez, A., Garret, M., Toulme, E., and Boue-Grabot, E.(2011). Cross-talk between P2X4 and gamma-aminobutyric acid, type Areceptors determines synaptic efficacy at a central synapse. J. Biol. Chem. 286,19993–20004. doi: 10.1074/jbc.M111.231324

Joers, V., Tansey, M. G., Mulas, G., and Carta, A. R. (2016). Microglial phenotypesin Parkinson’s disease and animal models of the disease. Prog. Neurobiol. [Epubahead of print].

Jones, C. A., Chessell, I. P., Simon, J., Barnard, E. A., Miller, K. J., Michel, A. D.,et al. (2000). Functional characterization of the P2X4 receptor orthologues. Br.J. Pharmacol. 129, 388–394. doi: 10.1038/sj.bjp.0703059

Kawate, T., Michel, J. C., Birdsong, W. T., and Gouaux, E. (2009). Crystal structureof the ATP-gated P2X4 ion channel in the closed state. Nature 460, 592–598.doi: 10.1038/nature08198

Kerr, B. J., Bradbury, E. J., Bennett, D. L., Trivedi, P. M., Dassan, P., French, J.,et al. (1999). Brain-derived neurotrophic factor modulates nociceptive sensoryinputs and NMDA-evoked responses in the rat spinal cord. J. Neurosci. 19,5138–5148.

Kettenmann, H., Hanisch, U. K., Noda, M., and Verkhratsky, A. (2011). Physiologyof microglia. Physiol. Rev. 91, 461–553. doi: 10.1152/physrev.00011.2010

Khakh, B. S., Bao, X. R., Labarca, C., and Lester, H. A. (1999a). Neuronal P2Xtransmitter-gated cation channels change their ion selectivity in seconds. Nat.Neurosci. 2, 322–330.

Khakh, B. S., Proctor, W. R., Dunwiddie, T. V., Labarca, C., and Lester, H. A.(1999b). Allosteric control of gating and kinetics at P2X4 receptor channels.J. Neurosci. 19, 7289–7299.

Khakh, B. S., and North, R. A. (2006). P2X receptors as cell-surface ATP sensors inhealth and disease. Nature 442, 527–532. doi: 10.1038/nature04886

Khakh, B. S., and North, R. A. (2012). Neuromodulation by extracellular ATP andP2X receptors in the CNS. Neuron 76, 51–69. doi: 10.1016/j.neuron.2012.09.024

Khakh, B. S., Zhou, X., Sydes, J., Galligan, J. J., and Lester, H. A. (2000). State-dependent cross-inhibition between transmitter-gated cation channels. Nature406, 405–410. doi: 10.1038/35019066

Khoja, S., Shah, V., Garcia, D., Asatryan, L., Jakowec, M. W., and Davies,D. L. (2016). Role of purinergic P2X4 receptors in regulating striataldopamine homeostasis and dependent behaviors. J. Neurochem. 139, 134–148.doi: 10.1111/jnc.13734

Kimpel, M. W., Strother, W. N., Mcclintick, J. N., Carr, L. G., Liang, T., Edenberg,H. J., et al. (2007). Functional gene expression differences between inbredalcohol-preferring and –non-preferring rats in five brain regions. Alcohol 41,95–132. doi: 10.1016/j.alcohol.2007.03.003

Kobayashi, K., Fukuoka, T., Yamanaka, H., Dai, Y., Obata, K., Tokunaga, A., et al.(2005). Differential expression patterns of mRNAs for P2X receptor subunits in

Frontiers in Pharmacology | www.frontiersin.org 12 May 2017 | Volume 8 | Article 291

fphar-08-00291 May 22, 2017 Time: 17:37 # 13

Stokes et al. P2X4 Function and Pharmacology

neurochemically characterized dorsal root ganglion neurons in the rat. J. Comp.Neurol. 481, 377–390. doi: 10.1002/cne.20393

Krause, R. M., Buisson, B., Bertrand, S., Corringer, P.-J., Galzi, J.-L., Changeux,J.-P., et al. (1998). Ivermectin: a positive allosteric effector of the α7 neuronalnicotinic acetylcholine receptor. Mol. Pharmacol. 53, 283.

Krušek, J., and Zemková, H. (1994). Effect of ivermectin on γ-aminobutyric acid-induced chloride currents in mouse hippocampal embryonic neurones. Eur. J.Pharmacol. 259, 121–128. doi: 10.1016/0014-2999(94)90500-2

Kukley, M., Barden, J. A., Steinhäuser, C., and Jabs, R. (2001). Distribution ofP2X receptors on astrocytes in juvenile rat hippocampus. Glia 36, 11–21.doi: 10.1002/glia.1091

Laing, R., Gillan, V., and Devaney, E. (2017). Ivermectin – Old Drug, New Tricks?Trends Parasitol. [Epub ahead of print].

Lalo, U., Pankratov, Y., Wichert, S. P., Rossner, M. J., North, R. A., Kirchhoff, F.,et al. (2008). P2X1 and P2X5 subunits form the functional P2X receptorin mouse cortical astrocytes. J. Neurosci. 28, 5473–5480. doi: 10.1523/JNEUROSCI.1149-08.2008

Lalo, U., Verkhratsky, A., and Pankratov, Y. (2007). Ivermectin potentiates ATP-induced ion currents in cortical neurones: evidence for functional expression ofP2X4 receptors? Neurosci. Lett. 421, 158–162. doi: 10.1016/j.neulet.2007.03.078

Le, K. T., Babinski, K., and Seguela, P. (1998). Central P2X4 and P2X6 channelsubunits coassemble into a novel heteromeric ATP receptor. J. Neurosci. 18,7152–7159.

Lê, K. T., Villeneuve, P., Ramjaun, A. R., Mcpherson, P. S., Beaudet, A.,and Séguéla, P. (1998). Sensory presynaptic and widespread somatodendriticimmunolocalization of central ionotropic P2X ATP receptors. Neuroscience 83,177–190. doi: 10.1016/S0306-4522(97)00365-5

Li, F., Wang, L., Li, J. W., Gong, M., He, L., Feng, R., et al. (2011). Hypoxia inducedamoeboid microglial cell activation in postnatal rat brain is mediated by ATPreceptor P2X4. BMC Neurosci. 12:111. doi: 10.1186/1471-2202-12-111

Li, J., and Fountain, S. J. (2012). Fluvastatin suppresses native and recombinanthuman P2X4 receptor function. Purinergic Signal. 8, 311–316. doi: 10.1007/s11302-011-9289-9

Li, M., Toombes, G. E. S., Silberberg, S. D., and Swartz, K. J. (2015). Physical basis ofapparent pore dilation of ATP-activated P2X receptor channels. Nat. Neurosci.18, 1577–1583. doi: 10.1038/nn.4120

Luo, J., Yin, G.-F., Gu, Y.-Z., Liu, Y., Dai, J.-P., Li, C., et al. (2006). Characterizationof three types of ATP-activated current in relation to P2X subunits in rattrigeminal ganglion neurons. Brain Res. 1115, 9–15. doi: 10.1016/j.brainres.2006.07.084

Mapplebeck, J. C., Beggs, S., and Salter, M. W. (2016). Sex differences in pain:a tale of two immune cells. Pain 157(Suppl. 1), S2–S6. doi: 10.1097/j.pain.0000000000000389

Masuda, T., Iwamoto, S., Yoshinaga, R., Tozaki-Saitoh, H., Nishiyama, A.,Mak, T. W., et al. (2014). Transcription factor IRF5 drives P2X4R+-reactivemicroglia gating neuropathic pain. Nat. Commun. 5:3771. doi: 10.1038/ncomms4771

Matsumura, Y., Yamashita, T., Sasaki, A., Nakata, E., Kohno, K., Masuda, T., et al.(2016). A novel P2X4 receptor-selective antagonist produces anti-allodyniceffect in a mouse model of herpetic pain. Sci. Rep. 6:32461. doi: 10.1038/srep32461

Miklavc, P., Thompson, K. E., and Frick, M. (2013). A new role for P2X4receptors as modulators of lung surfactant secretion. Front. Cell. Neurosci 7:171.doi: 10.3389/fncel.2013.00171

Miller, K. J., Michel, A. D., Chessell, I. P., and Humphrey, P. P. (1998). Cibacronblue allosterically modulates the rat P2X4 receptor. Neuropharmacology 37,1579–1586. doi: 10.1016/S0028-3908(98)00153-1

Nagata, K., Imai, T., Yamashita, T., Tsuda, M., Tozaki-Saitoh, H., and Inoue, K.(2009). Antidepressants inhibit P2X4 receptor function: a possible involvementin neuropathic pain relief. Mol. Pain 5:20. doi: 10.1186/1744-8069-5-20

Nasu-Tada, K., Koizumi, S., Tsuda, M., Kunifusa, E., and Inoue, K. (2006). Possibleinvolvement of increase in spinal fibronectin following peripheral nerve injuryin upregulation of microglial P2X4, a key molecule for mechanical allodynia.Glia 53, 769–775. doi: 10.1002/glia.20339

Neher, E., and Sakaba, T. (2008). Multiple roles of calcium ions in the regulationof neurotransmitter release. Neuron 59, 861–872. doi: 10.1016/j.neuron.2008.08.019

Nicke, A., Kerschensteiner, D., and Soto, F. (2005). Biochemical and functionalevidence for heteromeric assembly of P2X1 and P2X4 subunits. J. Neurochem.92, 925–933. doi: 10.1111/j.1471-4159.2004.02939.x

Nörenberg, W., Sobottka, H., Hempel, C., Plötz, T., Fischer, W., Schmalzing, G.,et al. (2012). Positive allosteric modulation by ivermectin of human but notmurine P2X7 receptors. Br. J. Pharmacol. 167, 48–66. doi: 10.1111/j.1476-5381.2012.01987.x

North, R. A. (2002). Molecular physiology of P2X receptors. Physiol. Rev. 82,1013–1067. doi: 10.1152/physrev.00015.2002

Oesterle, A., Laufs, U., and Liao, J. K. (2017). Pleiotropic effects of statins onthe cardiovascular system. Circ. Res. 120, 229. doi: 10.1161/CIRCRESAHA.116.308537

Ostrovskaya, O., Asatryan, L., Wyatt, L., Popova, M., Li, K., Peoples, R. W., et al.(2011). Ethanol is a fast channel inhibitor of P2X4 receptors. J. Pharmacol. Exp.Ther. 337, 171–179. doi: 10.1124/jpet.110.176990

Ousingsawat, J., Wanitchakool, P., Kmit, A., Romao, A. M., Jantarajit, W.,Schreiber, R., et al. (2015). Anoctamin 6 mediates effects essential for innateimmunity downstream of P2X7 receptors in macrophages. Nat. Commun.6:6245. doi: 10.1038/ncomms7245

Ozaki, T., Muramatsu, R., Sasai, M., Yamamoto, M., Kubota, Y., Fujinaka, T.,et al. (2016). The P2X4 receptor is required for neuroprotection via ischemicpreconditioning. Sci. Rep. 6, 25893. doi: 10.1038/srep25893

Pelegrin, P., and Surprenant, A. (2006). Pannexin-1 mediates large pore formationand interleukin-1β release by the ATP-gated P2X&lt;sub&gt;7&lt;/sub&gt;receptor. EMBO J. 25, 5071. doi: 10.1038/sj.emboj.7601378

Popova, M., Asatryan, L., Ostrovskaya, O., Wyatt, L. R., Li, K., Alkana, R. L.,et al. (2010). A point mutation in the ectodomain-transmembrane 2 interfaceeliminates the inhibitory effects of ethanol in P2X4 receptors. J. Neurochem.112, 307–317. doi: 10.1111/j.1471-4159.2009.06460.x

Popova, M., Trudell, J., Li, K., Alkana, R., Davies, D., and Asatryan, L. (2013).Tryptophan 46 is a site for ethanol and ivermectin action in P2X4 receptors.Purinergic Signal 9, 621–632. doi: 10.1007/s11302-013-9373-4

Potula, R., Haorah, J., Knipe, B., Leibhart, J., Chrastil, J., Heilman, D., et al. (2006).Alcohol abuse enhances neuroinflammation and impairs immune responsesin an animal model of human immunodeficiency virus-1 encephalitis. Am. J.Pathol. 168, 1335–1344. doi: 10.2353/ajpath.2006.051181

Priel, A., and Silberberg, S. D. (2004). Mechanism of ivermectin facilitation ofhuman P2X4 receptor channels. J. Gen. Physiol. 123, 281–293. doi: 10.1085/jgp.200308986

Qureshi, O. S., Paramasivam, A., Yu, J. C., and Murrell-Lagnado, R. D. (2007).Regulation of P2X4 receptors by lysosomal targeting, glycan protection andexocytosis. J. Cell Sci. 120, 3838–3849. doi: 10.1242/jcs.010348

Ransohoff, R. M. (2016). How neuroinflammation contributes toneurodegeneration. Science 353, 777–783. doi: 10.1126/science.aag2590

Raouf, R., Chabot-Dore, A. J., Ase, A. R., Blais, D., and Seguela, P. (2007).Differential regulation of microglial P2X4 and P2X7 ATP receptors followingLPS-induced activation. Neuropharmacology 53, 496–504. doi: 10.1016/j.neuropharm.2007.06.010

Robinson, L. E., and Murrell-Lagnado, R. D. (2013). The trafficking and targetingof P2X receptors. Front. Cell. Neurosci. 7:233. doi: 10.3389/fncel.2013.00233

Rokic, M. B., Stojilkovic, S. S., Vavra, V., Kuzyk, P., Tvrdonova, V., andZemkova, H. (2013). Multiple roles of the extracellular vestibule amino acidresidues in the function of the rat P2X4 receptor. PLoS ONE 8:e59411.doi: 10.1371/journal.pone.0059411

Royle, S. J., Qureshi, O. S., Bobanovic, L. K., Evans, P. R., Owen, D. J., andMurrell-Lagnado, R. D. (2005). Non-canonical YXXGPhi endocytic motifs:recognition by AP2 and preferential utilization in P2X4 receptors. J. Cell Sci.118, 3073–3080. doi: 10.1242/jcs.02451

Rubio, M. E., and Soto, F. (2001). Distinct localization of P2X receptors atexcitatory postsynaptic specializations. J. Neurosci. 21, 641–653.

Saijo, K., and Glass, C. K. (2011). Microglial cell origin and phenotypes in healthand disease. Nat. Rev. Immunol. 11, 775–787. doi: 10.1038/nri3086

Samways, D. S., Khakh, B. S., and Egan, T. M. (2012). Allosteric modulation ofCa2+ flux in ligand-gated cation channel (P2X4) by actions on lateral portals.J. Biol. Chem. 287, 7594–7602. doi: 10.1074/jbc.M111.322461

Samways, D. S. K., Khakh, B. S., Dutertre, S., and Egan, T. M. (2011). Preferentialuse of unobstructed lateral portals as the access route to the pore of human

Frontiers in Pharmacology | www.frontiersin.org 13 May 2017 | Volume 8 | Article 291

fphar-08-00291 May 22, 2017 Time: 17:37 # 14

Stokes et al. P2X4 Function and Pharmacology

ATP-gated ion channels (P2X receptors). Proc. Natl. Acad. Sci. U.S.A. 108,13800–13805. doi: 10.1073/pnas.1017550108

Sathanoori, R., Sward, K., Olde, B., and Erlinge, D. (2015). The ATP ReceptorsP2X7 and P2X4 modulate high glucose and palmitate-induced inflammatoryresponses in endothelial cells. PLoS ONE 10:e0125111. doi: 10.1371/journal.pone.0125111

Saul, A., Hausmann, R., Kless, A., and Nicke, A. (2013). Heteromeric assembly ofP2X subunits. Front. Cell. Neurosci. 7:250. doi: 10.3389/fncel.2013.00250

Schwab, J. M., Guo, L., and Schluesener, H. J. (2005). Spinal cord injury inducesearly and persistent lesional P2X4 receptor expression. J. Neuroimmunol. 163,185–189. doi: 10.1016/j.jneuroim.2005.02.016

Shinozaki, Y., Sumitomo, K., Tsuda, M., Koizumi, S., Inoue, K., and Torimitsu, K.(2009). Direct Observation of ATP-induced conformational changes in singleP2X4 receptors. PLoS Biol. 7:e1000103. doi: 10.1371/journal.pbio.1000103

Shrivastava, A. N., Triller, A., and Sieghart, W. (2011). GABA(A) receptors:post-synaptic co-localization and cross-talk with other receptors. Front. Cell.Neurosci. 5:7. doi: 10.3389/fncel.2011.00007

Silberberg, S. D., Li, M., and Swartz, K. J. (2007). Ivermectin interaction withtransmembrane helices reveals widespread rearrangements during opening ofP2X receptor channels. Neuron 54, 263–274. doi: 10.1016/j.neuron.2007.03.020

Sim, J. A., Chaumont, S., Jo, J., Ulmann, L., Young, M. T., Cho, K., et al.(2006). Altered hippocampal synaptic potentiation in P2X4 knock-out mice.J. Neurosci. 26, 9006–9009. doi: 10.1523/JNEUROSCI.2370-06.2006

Sim, J. A., and North, R. A. (2010). Amitriptyline does not block the action ofATP at human P2X4 receptor. Br. J. Pharmacol. 160, 88–92. doi: 10.1111/j.1476-5381.2010.00683.x

Sorge, R. E., Mapplebeck, J. C., Rosen, S., Beggs, S., Taves, S., Alexander, J. K.,et al. (2015). Different immune cells mediate mechanical pain hypersensitivityin male and female mice. Nat. Neurosci. 18, 1081–1083. doi: 10.1038/nn.4053

Soto, F., Garcia-Guzman, M., Gomez-Hernandez, J. M., Hollmann, M.,Karschin, C., and Stuhmer, W. (1996a). P2X4: an ATP-activated ionotropicreceptor cloned from rat brain. Proc. Natl. Acad. Sci. U.S.A. 93, 3684–3688.

Soto, F., Garcia-Guzman, M., Karschin, C., and Stühmer, W. (1996b). Cloning andtissue distribution of a novel P2X receptor from rat brain. Biochem. Biophys.Res. Commun. 223, 456–460.

Stojilkovic, S. S. (2009). Purinergic regulation of hypothalamopituitary functions.Trends Endocrinol. Metab. 20, 460–468. doi: 10.1016/j.tem.2009.05.005

Stojilkovic, S. S., and Zemkova, H. (2013). P2X receptor channels in endocrineglands, Wiley interdisciplinary reviews. Membr. Trans. Signal. 2, 173–180.doi: 10.1002/wmts.89

Stokes, L., Scurrah, K., Ellis, J. A., Cromer, B. A., Skarratt, K. K., Gu, B. J.,et al. (2011). A loss-of-function polymorphism in the human P2X4 receptoris associated with increased pulse pressure. Hypertension 58, 1086–1092.doi: 10.1161/HYPERTENSIONAHA.111.176180

Stokes, L., and Surprenant, A. (2009). Dynamic regulation of the P2X4 receptor inalveolar macrophages by phagocytosis and classical activation. Eur. J. Immunol.39, 986–995. doi: 10.1002/eji.200838818

Surprenant, A., and North, R. A. (2009). Signaling at purinergic P2X receptors.Annu. Rev. Physiol. 71, 333–359. doi: 10.1146/annurev.physiol.70.113006.100630

Tan, Y., Zhao, B., Zeng, Q.-C., Shi, C.-M., Zhao, F.-B., and Li, Z.-W. (2009).Characteristics of ATP-activated current in nodose ganglion neurons of rats.Neurosci. Lett. 459, 25–29. doi: 10.1016/j.neulet.2009.04.054

Tian, M., Abdelrahman, A., Weinhausen, S., Hinz, S., Weyer, S., Dosa, S.,et al. (2014). Carbamazepine derivatives with P2X4 receptor-blockingactivity. Bioorg. Med. Chem. 22, 1077–1088. doi: 10.1016/j.bmc.2013.12.035

Toulme, E., Garcia, A., Samways, D., Egan, T. M., Carson, M. J., and Khakh, B. S.(2010). P2X4 receptors in activated C8-B4 cells of cerebellar microglial origin.J. Gen. Physiol. 135, 333–353. doi: 10.1085/jgp.200910336

Toulme, E., and Khakh, B. S. (2012). Imaging P2X4 receptor lateral mobility inmicroglia: regulation by calcium and p38 MAPK. J. Biol. Chem. 287, 14734–14748. doi: 10.1074/jbc.M111.329334

Toulme, E., Soto, F., Garret, M., and Boue-Grabot, E. (2006). Functional propertiesof internalization-deficient P2X4 receptors reveal a novel mechanism ofligand-gated channel facilitation by ivermectin. Mol. Pharmacol. 69, 576–587.doi: 10.1124/mol.105.018812

Toyomitsu, E., Tsuda, M., Yamashita, T., Tozaki-Saitoh, H., Tanaka, Y., andInoue, K. (2012). CCL2 promotes P2X4 receptor trafficking to the cell surfaceof microglia. Purinergic Signal. 8, 301–310. doi: 10.1007/s11302-011-9288-x

Trang, T., Beggs, S., Wan, X., and Salter, M. W. (2009). P2X4-receptor-mediatedsynthesis and release of brain-derived neurotrophic factor in microglia isdependent on calcium and p38-mitogen-activated protein kinase activation.J. Neurosci. 29, 3518–3528. doi: 10.1523/JNEUROSCI.5714-08.2009

Tsuda, M. (2016). P2 receptors, microglial cytokines and chemokines, andneuropathic pain. J. Neurosci. Res. 95, 1319–1329. doi: 10.1002/jnr.23816

Tsuda, M., Kuboyama, K., Inoue, T., Nagata, K., Tozaki-Saitoh, H., and Inoue, K.(2009a). Behavioral phenotypes of mice lacking purinergic P2X4 receptors inacute and chronic pain assays. Mol. Pain 5, 28. doi: 10.1186/1744-8069-5-28

Tsuda, M., Masuda, T., Kitano, J., Shimoyama, H., Tozaki-Saitoh, H., and Inoue, K.(2009b). IFN-γ receptor signaling mediates spinal microglia activation drivingneuropathic pain. Proc. Natl. Acad. Sci. U.S.A. 106, 8032–8037. doi: 10.1073/pnas.0810420106

Tsuda, M., Masuda, T., Tozaki-Saitoh, H., and Inoue, K. (2013). P2X4 receptors andneuropathic pain. Front. Cell. Neurosci. 7:191. doi: 10.3389/fncel.2013.00191

Tsuda, M., Shigemoto-Mogami, Y., Koizumi, S., Mizokoshi, A., Kohsaka, S., Salter,M. W., et al. (2003). P2X4 receptors induced in spinal microglia gate tactileallodynia after nerve injury. Nature 424, 778–783. doi: 10.1038/nature01786

Tsuda, M., Toyomitsu, E., Komatsu, T., Masuda, T., Kunifusa, E., Nasu-Tada, K.,et al. (2008). Fibronectin/integrin system is involved in P2X4 receptorupregulation in the spinal cord and neuropathic pain after nerve injury. Glia56, 579–585. doi: 10.1002/glia.20641

Ulmann, L., Hatcher, J. P., Hughes, J. P., Chaumont, S., Green, P. J., Conquet, F.,et al. (2008). Up-regulation of P2X4 receptors in spinal microglia afterperipheral nerve injury mediates BDNF release and neuropathic pain.J. Neurosci. 28, 11263–11268. doi: 10.1523/JNEUROSCI.2308-08.2008

Ulmann, L., Hirbec, H., and Rassendren, F. (2010). P2X4 receptors mediate PGE2release by tissue-resident macrophages and initiate inflammatory pain. EMBOJ. 29, 2290–2300. doi: 10.1038/emboj.2010.126

Ulmann, L., Levavasseur, F., Avignone, E., Peyroutou, R., Hirbec, H., Audinat, E.,et al. (2013). Involvement of P2X4 receptors in hippocampal microglialactivation after status epilepticus. Glia 61, 1306–1319. doi: 10.1002/glia.22516

Vavra, V., Bhattacharya, A., and Zemkova, H. (2011). Facilitation of glutamate andGABA release by P2X receptor activation in supraoptic neurons from freshlyisolated rat brain slices. Neuroscience 188, 1–12. doi: 10.1016/j.neuroscience.2011.04.067

Vazquez-Villoldo, N., Domercq, M., Martin, A., Llop, J., Gomez-Vallejo, V., andMatute, C. (2014). P2X4 receptors control the fate and survival of activatedmicroglia. Glia 62, 171–184. doi: 10.1002/glia.22596

Virginio, C., Robertson, G., Surprenant, A., and North, R. A. (1998).Trinitrophenyl-substituted nucleotides are potent antagonists selective forP2X1, P2X3 & heteromeric P2X2/3 receptors. Mol. Pharmacol. 53, 969–973.

Wes, P. D., Sayed, F. A., Bard, F., and Gan, L. (2016). Targeting microglia for thetreatment of Alzheimer’s Disease. Glia 64, 1710–1732. doi: 10.1002/glia.22988

Wheeler-Schilling, T. H., Marquordt, K., Kohler, K., Guenther, E., and Jabs, R.(2001). Identification of purinergic receptors in retinal ganglion cells. Mol.Brain Res. 92, 177–180. doi: 10.1016/S0169-328X(01)00160-7

Wildman, S. S., King, B. F., and Burnstock, G. (1999). Modulation of ATP-responses at recombinant rP2X(4) receptors by extracellular pH and zinc. Br.J. Pharmacol. 126, 762–768. doi: 10.1038/sj.bjp.0702325

Wixey, J. A., Reinebrant, H. E., Carty, M. L., and Buller, K. M. (2009). DelayedP2X4R expression after hypoxia–ischemia is associated with microglia in theimmature rat brain. J. Neuroimmunol. 212, 35–43. doi: 10.1016/j.jneuroim.2009.04.016

Wyatt, L. R., Finn, D. A., Khoja, S., Yardley, M. M., Asatryan, L., Alkana, R. L.,et al. (2014). Contribution of P2X4 receptors to ethanol intake in male C57BL/6mice. Neurochem. Res. 39, 1127–1139. doi: 10.1007/s11064-014-1271-9

Wyatt, L. R., Godar, S. C., Khoja, S., Jakowec, M. W., Alkana, R. L., Bortolato, M.,et al. (2013). Sociocommunicative and sensorimotor impairments in maleP2X4-deficient mice. Neuropsychopharmacology 38, 1993–2002. doi: 10.1038/npp.2013.98

Xiong, K., Hu, X. Q., Stewart, R. R., Weight, F. F., and Li, C. (2005). The mechanismby which ethanol inhibits rat P2X4 receptors is altered by mutation of histidine241. Br. J. Pharmacol. 145, 576–586. doi: 10.1038/sj.bjp.0706192

Frontiers in Pharmacology | www.frontiersin.org 14 May 2017 | Volume 8 | Article 291

fphar-08-00291 May 22, 2017 Time: 17:37 # 15

Stokes et al. P2X4 Function and Pharmacology

Xu, J., Chai, H., Ehinger, K., Egan, T. M., Srinivasan, R., Frick, M., et al. (2014).Imaging P2X4 receptor subcellular distribution, trafficking, and regulationusing P2X4-pHluorin. J. Gen. Physiol. 144, 81–104. doi: 10.1085/jgp.201411169

Yamamoto, K., Korenaga, R., Kamiya, A., and Ando, J. (2000). Fluid shear stressactivates Ca(2+) influx into human endothelial cells via P2X4 purinoceptors.Circ. Res. 87, 385–391. doi: 10.1161/01.RES.87.5.385

Yamamoto, K., Sokabe, T., Matsumoto, T., Yoshimura, K., Shibata, M., Ohura, N.,et al. (2006). Impaired flow-dependent control of vascular tone and remodelingin P2X4-deficient mice. Nat. Med. 12, 133–137. doi: 10.1038/nm1338

Yamashita, T., Yamamoto, S., Zhang, J., Kometani, M., Tomiyama, D.,Kohno, K., et al. (2016). Duloxetine inhibits microglial P2X4 receptor functionand alleviates neuropathic pain after peripheral nerve injury. PLoS ONE11:e0165189. doi: 10.1371/journal.pone.0165189

Yang, T., Shen, J. B., Yang, R., Redden, J., Dodge-Kafka, K., Grady, J., et al. (2014).Novel protective role of endogenous cardiac myocyte P2X4 receptors in heartfailure. Circ. Heart Fail. 7, 510–518. doi: 10.1161/CIRCHEARTFAILURE.113.001023

Yardley, M. M., Wyatt, L., Khoja, S., Asatryan, L., Ramaker, M. J., Finn, D. A.,et al. (2012). Ivermectin reduces alcohol intake and preference in mice.Neuropharmacology 63, 190–201. doi: 10.1016/j.neuropharm.2012.03.014

Zarei, M., Sabetkasaei, M., and Moini Zanjani, T. (2014). Paroxetine attenuates thedevelopment and existing pain in a rat model of neurophatic pain. Ir. Biomed.J. 18, 94–100.

Zemkova, H., Kucka, M., Li, S., Gonzalez-Iglesias, A. E., Tomic, M., and Stojilkovic,S. S. (2010). Characterization of purinergic P2X4 receptor channels expressed inanterior pituitary cells. Am. J. Physiol. Endocrinol. Metab. 298, E644–E651.doi:10.1152/ajpendo.00558.2009

Conflict of Interest Statement: The authors declare that the research wasconducted in the absence of any commercial or financial relationships that couldbe construed as a potential conflict of interest.

Copyright © 2017 Stokes, Layhadi, Bibic, Dhuna and Fountain. This is anopen-access article distributed under the terms of the Creative CommonsAttribution License (CC BY). The use, distribution or reproduction inother forums is permitted, provided the original author(s) or licensorare credited and that the original publication in this journal is cited,in accordance with accepted academic practice. No use, distributionor reproduction is permitted which does not comply with theseterms.

Frontiers in Pharmacology | www.frontiersin.org 15 May 2017 | Volume 8 | Article 291